Methods of regulating cannabinoid receptor activity-related disorders and diseases

ABSTRACT

This disclosure concerns the discovery of the use of fenoterol analogues for regulating cannabinoid (CB) receptor activity-related disorders and disease, such as dysregulated CB receptors, including treating a disorder or disease, such as a glioblastoma, hepatocellular carcinoma, liver cancer, colon cancer, and/or lung cancer, which is associated with altered cannabinoid receptor activity. In one example, the method includes administering to a subject having or at risk of developing a disorder or disease regulated by CB receptor activity an effective amount of a fenoterol analogue to reduce one or more symptoms associated with the disorder or disease regulated by CB receptor activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 15/225,643, filedAug. 1, 2016, which is a continuation of U.S. application Ser. No.14/403,516, filed Nov. 24, 2014, now abandoned, which is the U.S.National Stage of International Application No. PCT/US2013/042457, filedMay 23, 2013, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Provisional Patent ApplicationNos. 61/651,961, filed on May 25, 2012, and 61/789,629, filed on Mar.15, 2013, each of which is hereby incorporated by reference in itsentirety.

FIELD

The present disclosure relates to the field of cannabinoid receptorsand, in particular, to methods of regulating cannabinoid (CB) receptoractivity-related disorders and diseases, such as activating CBreceptors, including treating a disorder or disease, such as aglioblastoma, hepatocellular carcinoma, liver cancer, colon cancer,and/or lung cancer, which is associated with altered cannabinoidreceptor activity by administration of specific fenoterol analogues.

BACKGROUND

Cancer is the second leading cause of human death next to coronarydisease in the United States. Worldwide, millions of people die fromcancer every year. In the United States alone, as reported by theAmerican Cancer Society, cancer causes the death of well over ahalf-million people annually, with over 1.2 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise.Cancer is soon predicted to become the leading cause of death. Manytypes of cancers, including brain and liver cancers, have no effectiveclinical treatments.

SUMMARY

This disclosure concerns the discovery that specific fenoterol analoguesare cannabinoid (CB) receptor modulators and can be used to treat adisorder or disease such as a tumor, including, but not limited to, aglioblastoma or hepatocellular carcinoma that is associated with alteredCB receptor activity or expression (or both), such as altered expressionor activity (or both) of the GPR55 cannabinoid receptor. The inventorshave discovered that administration of specific fenoterol analoguesinhibits one or more signs or symptoms (such as tumor growth) associatedwith a tumor that expresses a CB receptor. Using this discovery, theinventors developed the disclosed methods of treating a CBreceptor-modulated disorder or disease, including treatment of a tumorexpressing a CB receptor; for example, a glioblastoma or hepatocellularcarcinoma expressing a CB receptor.

In some embodiments, the method includes administering to a subjecthaving or at risk of developing a disorder or disease regulated by CBreceptor activity an effective amount of a compound to reduce one ormore symptoms associated with the disorder or disease regulated by CBreceptor activity, wherein the compound has the formula

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl, aminocarbonyl (carbamoyl) or a combination thereof; R₄ is H or lower alkyl;R₅ is

wherein Y¹, Y² and Y³ independently are hydrogen, halogen,sulphur-containing moiety including SH, sulfoxides, sulphones,sulphanamides and related alkyl and aromatic substituted moieties, lower—OR₆ and —NR₇R₈; R₆ is H or lower alkyl; R₇ and R₈ independently arehydrogen, lower alkyl, alkoxy carbonyl, acyl or amino carbonyl andwherein the compound is optically active, thereby reducing the one ormore symptoms associated with the disorder or disease in the subjectregulated by CB receptor activity.

In some embodiments, administering comprises administering atherapeutically effective amount of a compound, wherein R₄ within thecompound is selected from methyl, ethyl, n-propyl, and isopropyl.

In some embodiments, administering comprises administering atherapeutically effective amount of a compound, wherein R₄ within thecompound is methyl.

In some embodiments, administering comprises administering atherapeutically effective amount of a compound, wherein R₆ within thecompound is methyl.

In some embodiments, administering comprises administering atherapeutically effective amount of a compound, wherein R₅ within thecompound is one of

In some embodiments, administering comprises administering atherapeutically effective amount of a compound, wherein R₁-R₃ within thecompound are hydrogen.

In some embodiments, administering comprises administering atherapeutically effective amount of(R,R′)-4′-methoxy-1-naphthylfenoterol (MNF), (R,S′)-MNF,(R,R′)-ethylMNF, (R,R′)-naphthylfenoterol (NF), (R,R′)-ethylNF,(R,S′)-NF and (R,R′)-4′-amino-1-naphthylfenoterol (aminoNF),(R,R′)-4′-hydroxy-1-naphthylfenoterol (hydroxyNF), or a combinationthereof.

In some embodiments, administering comprises administering atherapeutically effective amount of MNF, NF or a combination thereof.

In some embodiments, administering comprises administering atherapeutically effective amount of MNF.

In some embodiments, the method includes administering a therapeuticallyeffective amount of a pharmaceutical composition containing any of thedisclosed fenoterol analogues capable of regulating a CBreceptor-associated disorder or disease and a pharmaceuticallyacceptable carrier to treat the disorder or disease regulated by a CBreceptor, such as a glioblastoma or hepatocellular carcinoma expressingGPR55. For example, the disclosed (R,R′)-MNF, (R,S′)-MNF,(R,R′)-ethylMNF, (R,R′)-NF, (R,R′)-ethylNF, (R,S′)-NF and(R,R′)-aminoNF, (R,R′)-hydroxyNF, or a combination thereof are effectiveat treating a glioblastoma or hepatocellular carcinoma expressing a CBreceptor, such as a GPR55 expressing glioblastoma or hepatocellularcarcinoma. In some embodiments, the method further includes selecting asubject having or at risk of developing a disorder or disease regulatedby a CB receptor. For example, a subject is selected for treatment bydetermining that the disorder or tumor is associated with CB receptorexpression, such as GPR55 expression. In one particular example, themethod further includes selecting a subject with a disorder and/ordisease, which is not associated with altered β2-AR function. Forexample, the disorder or disease does not respond to a treatmenttargeting β2-AR activity. In further examples, the method includesadministering one or more therapeutic agents in addition to thefenoterol analogue or combination thereof. The methods can includeadministration of the one or more therapeutic agents separately,sequentially or concurrently, for example in a combined composition witha fenoterol analogue or combinations thereof.

In some embodiments, the method is for use in treating a tumorexpressing a CB-receptor. For example, the disorder or disease isselected from the group consisting of a primary brain tumor expressing aCB-receptor, a glioblastoma expressing a CB-receptor, a hepatocellularcarcinoma expressing a CB-receptor, colon cancer, liver cancer, and lungcancer.

In some embodiments, inhibiting one or more signs or symptoms associatedwith the disease or disorder comprises inhibiting cellular growth, suchas tumor and/or cancer cell growth, tumor volume or a combinationthereof.

In some embodiments, the method is used to treat a disorder or diseaseregulated by a CB receptor, which is GPR55, such as diabetes.

The foregoing and other features and advantages of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate responses of HepG2 cells exposed to β-agoniststimulation. FIG. 1A is a digital image of soluble extracts which wereprepared from HepG2 (lane 1) and 1321N1 (lane 2) cells maintained incomplete medium, and subjected to Western blot analysis. Cellularcontent in β2-adrenergic receptor (β2-AR) and β-actin was measured usingspecific primary antibodies. FIG. 1B is a bar graph illustratingincreases in cAMP accumulation in HepG2 cells with forskolin, but notwith (R)-isoproterenol (Iso) or (R,R′)-fenoterol (Fen). Data shown arefrom a single study conducted in quadruplicate. Error bars indicatemean±S.D. from a single study. FIG. 1C is a digital image of animmunoblot. Serum-starved HepG2 cells were incubated in the presence of(R)-isoproterenol (Iso; 1 μM) or (R,R′)-Fen (1 μM) for 5, 10 and 30minutes. Cell lysates were immunoblotted with antibodies againstphosphorylated (Ser473) and total Akt, as well as phosphorylated ERK1/2and total ERK2. The studies shown in FIGS. 1B and 1C were repeated twicewith comparable results. The migration of molecular mass markers (valuesin kilodaltons) is shown on the left of immunoblots.

FIGS. 2A-D illustrate the effects of (R)-isoproterenol, (R,R′)-Fen andderivatives on cell growth are cell-type specific. Serum-starved HepG2cells were incubated with vehicle or the indicated concentrations of(R)-isoproterenol (Iso), (R,R′)-Fen, (R,R′)-aminoFen (NH₂-fen) or(R,R′)-MNF for 24 hours, and levels of [³H]-thymidine incorporation wasmeasured. Representative dose-response curves are shown in FIGS. 2A and2B. HepG2 cells in serum-depleted medium and 1321N1 cells in completemedium were treated with compounds at 1 μM for 24 hours and thoseresults are shown in FIG. 2C. FIG. 2D illustrates the findings whenHepG2 and 1321N1 cells were incubated without (SFM) or with serum (CM)in the presence of the indicated concentrations of Iso or (R,R′)-Fen(fen). Quantification of percent change in [³H]-thymidine incorporationversus control are expressed as means±SE and represent results from twoto six independent studies, each performed in triplicate dishes. In mostinstances, error bars are smaller than the symbols.

FIGS. 3A-3D demonstrate that a β2-AR antagonist does not inhibit theanti-proliferative action of (R,R′)-MNF in HepG2 cells. Serum-depletedHepG2 cells were incubated with the indicated concentrations of theβ2-AR antagonist, ICI-118,551 (ICI), for 1 hour followed by the additionof vehicle (FIG. 3A), (R,R′)-Fen (FIG. 3B, left panel), or (R,R′)-MNF(FIG. 3B, right panel) for 24 hours, and levels of [³H]-thymidineincorporation were measured. Representative dose-response curves for(R,R′)-Fen and (R,R′)-MNF are shown FIG. 3B. FIGS. 3C and 3D are bargraphs illustrating quantification of percent change in [³H]-thymidineincorporation versus control expressed as means±SE and represent resultsfrom three independent studies, each performed in triplicate.

FIG. 4 illustrates (R,R′)-MNF increases the number of sub-G1 events inHepG2 cells. Serum-depleted HepG2 cells were harvested after 6-hour,12-hour or 24-hour treatment with vehicle, (R,R′)-Fen (1 μM) or(R,R′)-MNF (1 μM). Cells were fixed, stained, and analyzed for DNAcontent using flow cytometry. Representative DNA content analysis invarious phases of the cell cycle after 24-hour treatment with vehicle,(R,R′)-Fen, or (R,R′)-MNF are shown. The number of sub-G1 events, whichrepresents dead cells or cells in late-stage apoptosis, was quantifiedas a function of treatment duration using results from two independentstudies, each performed in duplicate (lower right panel). Data areexpressed as means±SE (n=4).

FIG. 5 illustrates the results of flow cytometry studies in which(R,R′)-MNF induced apoptosis in HepG2 cells. Serum-depleted HepG2 cellswere treated with vehicle, (R,R′)-Fen (1 μM), or (R,R′)-MNF (1 μM) for24 hours; stained with Annexin V and propidium iodide (PI); and thenanalyzed by flow cytometry. Representative profiles are shown. Thefraction of annexin V-positive HepG2 cells that were apoptotic wasquantitated using results from two independent studies, each performedin duplicate (lower right panel). Data are expressed as means±SE (n=4).

FIGS. 6A-6C illustrate the role of cannabinoid receptor activation inthe anti-proliferative action of (R,R′)-MNF in HepG2 cells. FIG. 6A,Total RNA was extracted from HepG2, 1321N1 and U87MG cells, and thenanalyzed semi-quantitatively by PCR. A non-template control (NTC) hasbeen included (lane 1). FIGS. 6B and 6C, Serum-depleted HepG2 cells wereincubated with the cannabinoid receptor agonist, WIN 55,212-2 (Win; 1μM), (FIG. 6B) or the cannabinoid receptor antagonists, AM251(1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide;1 μM) or AM630(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone,0.5 μM), (FIG. 6C) for 1 hour followed by the addition of vehicle,(R,R′)-Fen (0.5 μM), or (R,R′)-MNF (0.25 μM) for 24 hours.Quantification of percent change in [³H]-thymidine incorporation versuscontrol are expressed as means±SD and represent results from threeindependent studies, each performed in triplicate dishes.

FIG. 7 is a series of graphs illustrating selective inhibition of(R,R′)-Fen-mediated cell proliferation control by the β2-AR antagonist,ICI-118,551. HepG2, 1321N1 and U87MG cells were incubated with the β2-ARantagonist, ICI-118,551 (ICI, 1 μM), for 1 hour followed by the additionof vehicle, (R,R′)-Fen (0.5 μM), or (R,R′)-MNF (0.25 μM) for 24 hours,and levels of [³H]-thymidine incorporation was measured. Quantificationof percent change in [³H]-thymidine incorporation versus control areexpressed as means±SD and represent the results from three independentstudies, each performed in triplicate dishes.

FIGS. 8A-8D are bar graphs illustrating cannabinoid receptors play norole in cell proliferation control by (R,R′)-Fen. 1321N1 (FIG. 8A) andU87MG (FIG. 8C) cells were incubated with the cannabinoid receptoragonist, WIN 55,212-2 (Win; 0.5-1 μM) or the cannabinoid receptorantagonists, AM251 (0.5-1 μM) or AM630 (0.25-0.5 μM) (FIGS. 8B, 8D) for1 hour followed by the addition of vehicle, (R,R′)-Fen (0.5 μM), or(R,R′)-MNF (0.25 μM) for 24 hours. Quantification of percent change in[³H]-thymidine incorporation versus control is expressed as means±SD andrepresents the results from three independent studies, each performed intriplicate.

FIG. 9 illustrates cellular uptake of TocriFluor 1117 (T1117), afluorescent AM251 analog, in HepG2 cells. Cells were treated with(R,R′)-Fen (1 μM), (R,R′)-MNF (1 μM), or AM251 (10 μM) for 1 hourfollowed by addition of T1117 (0.1 μM). Cells were mounted on confocalmicroscope and maintained at 37° C. with CO₂. Images were captured every30 seconds for up to one hour.

FIG. 10 is a graph illustrating metabolic stability of (R,R′)-MNF onhuman and rat liver microsomes.

FIG. 11 is a bar graph illustrating cytochrome p450 (CYP) inhibition by(R,R′)-MNF. Human liver microsomes were incubated with 8 different CYPsubstrates and 1 or 10 μM MNF. MNF at 10 μM was determined to inhibitCYP2D6 and CYP3A4. The primary metabolite was determined to beO-demethylated-MNF.

FIGS. 12 and 13 are tracings illustrating plasma and brain tissueconcentrations of (R,R′)-MNF. FIG. 12 illustrates the analysis of aplasma sample obtained 30 minutes post IV administration of 10 mg/kgMNF. MNF and Gluc-MNF, in the insert of FIG. 12, are shown with nointerfering peaks being present in the control plasma matrix. FIG. 13illustrates the analysis of brain tissue obtained 30 minutes post IVadministration of 10 mg/kg MNF. The peak at 6.39 minutes is anunidentified compound present in control brain matrix (see insert ofFIG. 13).

FIG. 14 is a MNF concentration-time course in plasma and brain of maleSprague-Dawley rats after IV administration of 10 mg/kg MNF IV where n=3rats per time point (10 minutes to 24 hours in plasma and 10 to 60minutes in the brain). The MNF concentration in brain tissue was 200ng/mg tissue at 10 minutes after administration and peaked at 30 minutesat 800 ng/mg tissue. The relative distribution between the concentrationof MNF in blood (measured as ng/ml) and brain tissue (measured as ng/mgtissue) was 0.2 at 10 minutes and 1.0 at 30 minutes and 60 minutesreflecting an equivalent distribution between both the central andperipheral body compartments.

FIG. 15 is a series of bar graphs illustrating that MNF does not producesignificant negative effects on the central nervous system relative tothe effects produced by tetrahydrocannabinol (presented in FIG. 16).

FIG. 16 is a series of bar graphs illustrating the effects oftetrahydrocannabinol on central nervous system function.

FIG. 17 is a series of graphs illustrating [3H]-Thymidine incorporationin 96-well culture plates including Hep3B cells, PC3 cells or LN229cells.

FIGS. 18A, 18B and 18C illustrate MNF reduces proliferation of rat C6glioma cell line. FIG. 18A, Cell proliferation assay was performed inrat C6 glioma cells treated with increasing concentrations of MNF for 24hours followed by the addition of [³H]-thymidine for 16 hours. FIG. 18B,Cells were preincubated without or with the selective β2-AR blocker,ICI-118,551 (3 nM) for 30 minutes followed by the addition of vehicle or20 nM of MNF, (R,R′)-Fen or isoproterenol (ISO) for 24 hours. FIG. 18C,C6 glioma cells were pretreated in the presence or absence ofcannabinoid receptor inverse agonists, AM251 (0.5 and 1 μM) and AM630(0.5 μM), for 30 minutes followed by the addition of vehicle or 20 nMMNF for 24 hours. FIGS. 18B and 18C, [³H]-thymidine was determined aftera 16 hour-incubation. Bars represent the average±SD of a singleexperiment performed in triplicate wells. Similar results were obtainedin 2-3 independent experiments. FIG. 18D, Changes in cell morphologywere observed for C6 cells incubated with 20 nM MNF for 48 hours.

FIGS. 19A and 19B each include a graph illustrating MNF reduces tumorgrowth in vivo in a rat C6 glioma xenograft model. C6 tumor-bearingfemale nude mice were assigned randomly to either the vehicle or the MNFgroup. Treatment was given by injecting either 2 mg/kg MNF or citrate inPBS five days a week for 19 days. Tumor volume was monitored daily andmice were sacrificed on the day after the last treatment. FIG. 19A,Tumor volume over time is shown for MNF-treated animals compared withvehicle-treated, tumor-bearing mice (means±SEM; n=9-10). FIG. 19B, Theindividual results from two cohorts of animals are depicted as areaunder the plasma drug concentration-time curve (AUCs). The averageAUC±SEM for the vehicle group was 5450±518 (n=17) and MNF: 3217±265(n=19). The P value presented is for a two-tailed test.

FIGS. 20A-20D illustrate gene expression profiling in MNF-treated C6tumor-bearing mice. FIG. 20A, Gene clustering in the rat C6 xenografts:Principal component analysis (PCA) of rat C6 glioma xenograft treatedwith MNF vs. vehicle control. PCA was applied to the six independentsamples (3 MNF, 3 controls), and numbers refer to individual samplelabels. Analysis reveals clustering of samples into treatment groups.FIG. 20B, Cluster analysis of 100 gene sets altered by MNF treatment, ascompared to the control group, in cohort #1, cohort #2, and combinedcohorts (1+2). FIG. 20C, Zratios of selected genes of interest isdepicted, showing either up- or down-regulated expression after pairwisecomparison between MNF and the vehicle-treated group. FIG. 20D, TotalRNA from C6 xenograft tumors from MNF- and vehicle-treated mice wasextracted and analyzed for Sox4, Olig1, Galnt3, Cdkn3, Ccna2 and Bub1bmRNA levels by quantitative real-time PCR (mean±SD; n=5-6). Values werenormalized to GAPDH.

FIG. 20E demonstrates the negative impact of MNF on cyclin expression inC6 tumor xenografts. Lysates from tumor samples were separated bySDS-PAGE and Western blotting was carried out primary antibodies raisedagainst cyclin A and cyclin D. Membranes were reprobed for Hsp90, whichserved as a loading control. Upper panel, representative immunoblots;lower panels, scatter plot of data showing significant differences incyclin A and cyclin D1 expression between xenograft tumors from vehicleand MNF-treated mice. *, p<0.05; **, p<0.01 using two-tailed Student'st-test. FIG. 20F, C6 glioma cells were incubated with 20 nM MNF for 6and 24 hours after which lysates were prepared and immunoblotted forcyclin A and cyclin D1. Membranes were reprobed for β-actin, whichserved as a loading control. Upper panel, representative blots; lowerpanel, Bars represent densitometric quantification of each blot with thevalues in vehicle-treated cells set at 1.0. Bars represent means±SEMfrom three independent studies. a, b: significant difference betweengroups at P<0.01.

FIGS. 21A-21D illustrate rapid and saturable incorporation of T1117 inHepG2 cells. Cellular entry of T1117 was measured on a Zeiss 710confocal microscope with thermoregulated chamber system for live cellimaging. FIG. 21A, Serum-depleted HepG2 cells were incubated in thepresence of increasing concentrations of T1117 (2.5-100 nM). Plots ofsignal intensity versus time were generated from defined regions ofinterest (ROIs). Results are from 2-3 independent studies. FIG. 21B,Relative AUC data versus T1117 concentrations is shown, and theT1117-100 nM values were set at 1. FIG. 21C, The cellular incorporationof T1117 (100 nM) was carried out in the presence of a 100× molar excessof unlabeled AM251. Error bars indicate mean±S.D. (n=3 ROIs) from asingle study, which was repeated twice with comparable results. FIG.21D, Representative images at t=15 minutes are shown. Bar, 30 μm.

FIGS. 22A-22D illustrate a role for GPR55 in cellular incorporation ofT1117. FIG. 22A, HepG2 cells were transfected with siRNA oligos eitheragainst CB₁R, CB₂R or GPR55 or the non-silencing siRNA control for 48hours. Cells were maintained in serum-free medium for 3 hours, followedby the addition of T1117. Plots of signal intensity vs. time weregenerated from defined ROIs. Error bars indicate mean±S.D. of twoindependent studies, each performed with 3-4 ROIs. FIG. 22B, RelativeAUC data, and the control siRNA values were set at 1. FIG. 22C,Serum-depleted HepG2 cells were treated with vehicle (0.01% DMSO), AM630(1 μM), or WIN 55,212-2 (1 μM) for 1 hour followed by the addition ofT1117. Error bars indicate mean±S.D. (n=3 ROIs) from a single study,which was repeated twice with comparable results. FIG. 22D,Serum-depleted HepG2 cells were treated with vehicle (0.01% DMSO), CP55,940 (0.25 μM), or O-1602([5-methyl-4-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-1,3-benzenediol;0.25 μM) for 30 minutes followed by the addition of 10 nM T1117. Errorbars indicate mean±S.D. (n=3 ROIs) from a single study, which wasrepeated twice with comparable results.

FIGS. 23A-25D illustrate the effect of MNF on cellular uptake of T1117.Serum-depleted HepG2 (FIG. 23A, FIG. 23B) and PANC-1 (FIG. 18C, FIG.23D) cells were pretreated or not with MNF (1 μM) or AM251 (10 μM) for30 minutes followed by the addition of vehicle (0.1% DMSO), AM251 or MNFfor an additional 30 minutes. Cells were then incubated with 10 nMT1117. FIGS. 23A, C: Plots of signal intensity vs, time were generatedfrom defined ROIs. Error bars indicate mean±S.D. of two independentstudies, each performed with 3-4 ROIs. FIGS. 23B, D: Relative AUC data,and the DMSO values were set at 1.

FIGS. 24A and 24B show MNF impairs ligand-induced internalization ofGPR55. HEK293 cells stably transfected with 3×HA-tagged hGPR55 vectorwere serum-starved, and then incubated with anti-HA antibody in theabsence or presence of MNF (1 μM) for 45 minutes at 37° C. Afterextensive washing, O-1602 (5 μM) was added to the cells for 20 minutesat 37° C. to promote GPR55 internalization. Intact cells were fixed andthen incubated with anti-rabbit Alexa Fluor 488 antibody to label cellsurface GPR55. After a permeabilization step, anti-rabbit Alexa Fluor568 antibody was added to detect intracellular GPR55. Nuclei werecounterstained with DAPI. Scale bar=20 μm.

FIGS. 25A-25D illustrates impairment in GPR55 downstream signaling byMNF. Serum-depleted HepG2 (FIG. 25A, FIG. 25B) and PANC-1 (FIG. 25C,FIG. 25D) cells were pretreated or not in the presence of MNF (1 μM) for10 minutes followed by the addition of vehicle, O-1602 (2.5 and 10 μM),or 10% FBS for an additional 10 minutes. Cell lysates were prepared,separated by reducing SDS-PAGE gel electrophoresis and immunoblotted fortotal and phosphoactive forms of ERK. FIGS. 25A, C: Representativeimmunoblots; FIGS. 25B, D: phospho-ERK1/2 bands were normalized to totalERK2, and the O-1602-10 μM values were set at 1. Data are means of twoindependent dishes±range. The migration of molecular-mass markers(values in kilodaltons) is shown on the left of immunoblots.

FIGS. 26A-26C show MNF interferes with inducible changes in cellmorphology and expression of EGFR. Serum-starved HepG2 (FIG. 26A) andPANC-1 (FIG. 26B) cells were pre-incubated in the presence of DMSO(0.1%) or MNF (1 μM) for 30 minutes followed by the addition of AM251 (5μM) or O-1602 (5 μM) for 16 hours. Unstimulated PANC-1 cells displayedcuboidal morphology with and without MNF. White arrows show individualcells with filopodia. FIG. 26C, Cell lysates were prepared from similarstudies and immunoblotted for EGFR. The membranes were reprobed forHsp90, which served as a loading control.

FIGS. 27A-27D show MNF inhibits ligand-induced motility of HepG2 andPANC-1 cells in a wound-healing assay. Confluent HepG2 (FIG. 27A, FIG.27B) and PANC-1 (FIG. 27C, FIG. 27D) cells were subjected to scratchwound. Cells were incubated in the presence of DMSO (0.1%) or the GPR55agonist AM251 (1 μM) for 30 minutes, followed by the addition of MNF (1μM) where indicated. Images were captured at various time-points. FIGS.27A, C: The relative wound surface area was measured over time andplotted, and values at time 0 were set at 1. FIGS. 27B, D: The relativewound surface area of four independent observations at the 24-hour timepoint is plotted. *, *** P<0.05 and 0.001.

FIG. 28A provides the structure of 5′-TAMRA-3-phenylpropan-1-amine(TAMRA-PPA) and T1117.

FIG. 28B provides the mass spectrum of TAMPRA-PPA ion, m/z equals 548.0.

FIG. 28C provides a comparison of the cellular accumulation of T1117 (10nM) vs. TAMRA-PPA (20 nM) in serum-depleted HepG2 cells. Note theabsence of TAMRA-PPA incorporation in cells.

FIGS. 29A-29C are captured images of a representative wound-healingassay. Confluent HepG2 (FIG. 29A) and PANC-1 (FIG. 29B) cells weresubjected to scratch wound and treated as described above for FIGS.27A-27D. Similar profiles were obtained in four independent assays. FIG.29C, Confluent HepG2 and PANC-1 cells were subjected to scratch wound.Cells were incubated in the presence of the atypical cannabinoid O-1602(1 μM) for 30 minutes, followed by the addition of vehicle (DMSO, 0.1%)or MNF (1 μM). Images were captured at various time-points. The relativewound surface area at the 24-hour time point is plotted. ** P<0.01.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on Sep. 12, 2018, 2.17 KB, which is incorporated byreference herein.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

I. Introduction

Disclosed herein is the finding that specific fenoterol analogues, suchas MNF, inhibit the growth of various types of tumor cells, includingglioblastoma tumor cells, hepatocellular carcinoma cells, colon cancercells, lung cancer cells, and liver cancer cells. In particular, theinventors performed a series of studies to characterize fenoterolanalogues and determine their possible therapeutic activities. MNF wasobserved to inhibit the growth of human-derived hepatocellular carcinomacells (HepG2) and human-(U87MG) and rat-(C6) derived glioblastoma cellsusing in vitro incubation and in vivo in flank implanted C6 xenograft innude mice. The results were unexpected as MNF is a β2-AR agonist andthis class of compounds had been shown to increase cellular growth inHepG2 cells. Binding and functional studies were performed whichrevealed that MNF acts as an inhibitor of the GPR55 cannabinoid receptorand, as such, represents one of the first potential drugs directed atthis target. Initial pK studies demonstrated that the compound crossesthe blood brain barrier and initial toxicity studies indicated that thedrug had little off-target effects. The β2-AR agonist properties were apositive indication and suggest that MNF may have cardio-protectiveeffects. MNF was also found to be capable of significantly inhibitingadditional types of tumor cell growth, including, but not limited to,colon cancer cells, lung cancer cells and liver cancer cells. Further,additional fenoterol compounds, such as (R,R′)-1-naphthylfenoterol (NF),were found to inhibit hepatocellular carcinoma cell growth. Thus, theessence of the discovery is the identification of a new class ofcompounds that can be used to treat CB receptor related disorders anddiseases, and in particular GRP55-related disorders and diseases,including brain and liver cancers for which there are no currenteffective treatments. Based upon these findings, disclosed are methodsof regulating CB receptor activity and treating disorders and diseasesmodulated by CB receptor activity or expression (or both), such as GRP55activity or expression (or both).

II. Abbreviations and Terms Abbreviations

-   -   AKAP: A-kinase anchoring protein    -   AM251:        1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide    -   AM630:        1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole    -   AR: adrenergic receptor    -   BBB: blood brain barrier    -   β2-AR: β2-adrenergic receptor    -   CB: cannabinoid    -   ERK: extracellular regulated kinase    -   Fen: fenoterol    -   GPR55: G protein-coupled receptor 55    -   GPCR: G protein-coupled receptor    -   HPLC: high performance liquid chromatography    -   IAM-PC: immobilized artificial membrane chromatographic support    -   ICI 118,551:        3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol    -   ICYP: [¹²⁵I]cyanopindolol    -   IP: intraperitoneal    -   IV: intravenous    -   MNF: 4-methoxy-1-naphthylfenoterol    -   NF: naphthylfenoterol    -   OGTT: oral glucose tolerance test    -   UV: ultraviolet

Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the disclosed subject matter belongs.Definitions of common terms in chemistry may be found in The McGraw-HillDictionary of Chemical Terms, 1985, and The Condensed ChemicalDictionary, 1981.

Except as otherwise noted, any quantitative values are approximatewhether the word “about” or “approximately” or the like are stated ornot. The materials, methods, and examples described herein areillustrative only and not intended to be limiting. Any molecular weightor molecular mass values are approximate and are provided only fordescription. Except as otherwise noted, the methods and techniques ofthe present invention are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See, e.g., Loudon, Organic Chemistry, FourthEdition, New York: Oxford University Press, 2002, pp. 360-361,1084-1085; Smith and March, March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience,2001; or Vogel, A Textbook of Practical Organic Chemistry, IncludingQualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.

In order to facilitate review of the various embodiments disclosedherein, the following explanations of specific terms are provided:

Acyl: A group of the formula RC(O)— wherein R is an organic group.

Acyloxy: A group having the structure —OC(O)R, where R may be anoptionally substituted alkyl or optionally substituted aryl. “Loweracyloxy” groups are those where R contains from 1 to 10 (such as from 1to 6) carbon atoms.

Administration: To provide or give a subject a composition, such as apharmaceutical composition including one or more fenoterol analogues byany effective route. Exemplary routes of administration include, but arenot limited to, injection (such as subcutaneous, intramuscular,intradermal, intraperitoneal (IP), and intravenous (IV)), oral,sublingual, rectal, transdermal, intranasal, vaginal and inhalationroutes.

Alkoxy: A radical (or substituent) having the structure —O—R, where R isa substituted or unsubstituted alkyl. Methoxy (—OCH₃) is an exemplaryalkoxy group. In a substituted alkoxy, R is alkyl substituted with anon-interfering substituent. “Thioalkoxy” refers to —S—R, where R issubstituted or unsubstituted alkyl. “Haloalkyloxy” means a radical —ORwhere R is a haloalkyl.

Alkoxy carbonyl: A group of the formula —C(O)OR, where R may be anoptionally substituted alkyl or optionally substituted aryl. “Loweralkoxy carbonyl” groups are those where R contains from 1 to 10 (such asfrom 1 to 6) carbon atoms.

Alkyl: An acyclic, saturated, branched- or straight-chain hydrocarbonradical, which, unless expressly stated otherwise, contains from one tofifteen carbon atoms; for example, from one to ten, from one to six, orfrom one to four carbon atoms. This term includes, for example, groupssuch as methyl, ethyl, n-propyl, isopropyl, isobutyl, t-butyl, pentyl,heptyl, octyl, nonyl, decyl, or dodecyl. The term “lower alkyl” refersto an alkyl group containing from one to ten carbon atoms. Unlessexpressly referred to as an “unsubstituted alkyl,” alkyl groups caneither be unsubstituted or substituted. An alkyl group can besubstituted with one or more substituents (for example, up to twosubstituents for each methylene carbon in an alkyl chain). Exemplaryalkyl substituents include, for instance, amino groups, amide,sulfonamide, halogen, cyano, carboxy, hydroxy, mercapto,trifluoromethyl, alkyl, alkoxy (such as methoxy), alkylthio, thioalkoxy,arylalkyl, heteroaryl, alkylamino, dialkylamino, alkylsulfano, keto, orother functionality.

Amino carbonyl (carbamoyl): A group of the formula C(O)N(R)R′, wherein Rand R′ are independently of each other hydrogen or a lower alkyl group.

Anti-diabetic agent: A chemical or pharmaceutical anti-hyperglycemicagent or drug capable of treating diabetes, including, but not limitedto agents for alleviating the symptoms associated with type 2 diabetesor slowing the progression or onset of type 2 diabetes. Anti-diabeticagents are generally categorized into six classes: biguanides;thiazolidinediones; sulfonylureas; inhibitors of carbohydrateabsorption; fatty acid oxidase inhibitors and anti-lipolytic drugs; andweight-loss agents. The anti-diabetic agents include those agentsdisclosed in Diabetes Care, 22(4):623-634 (1999), herein incorporated byreference. One common class of anti-diabetic agents is thesulfonylureas, which are believed to increase secretion of insulin,decrease hepatic gluconeogenesis, and increase insulin receptorsensitivity. Another class of anti-diabetic agents is the biguanideantihyperglycemics, which decrease hepatic glucose production andintestinal absorption, and increase peripheral glucose uptake andutilization, without inducing hyperinsulinemia. In some examples, ananti-diabetic agent is a disclosed fenoterol analogue capable ofmodulating a CB receptor activity, such as GPR55 activity.

Astrocytoma: A tumor of the brain that originates in astrocytes. Anastrocytoma is an example of a primary tumor. Astrocytomas are the mostcommon glioma, and can occur in most parts of the brain and occasionallyin the spinal cord. However, astrocytomas are most commonly found in thecerebrum. In one example, an astrocytoma is inhibited by administeringto a subject a therapeutic effective amount of fenoterol, a fenoterolanalogue or a combination thereof, thereby inhibiting astrocytomagrowth.

β2-adrenergic receptor (β2-AR): A subtype of adrenergic receptors thatare members of the G-protein coupled receptor family. β2-AR subtype isinvolved in respiratory diseases, cardiovascular diseases, prematurelabor and, as disclosed herein, tumor development. Increased expressionof β2-ARs can serve as therapeutic targets. Currently, a number of drugse.g., albuterol, formoterol, isoproterenol, or salmeterol have β2-ARagonist activities. As disclosed herein, fenoterol and fenoterolanalogues are β2-AR agonists.

Blood-brain barrier (BBB): The barrier formed by epithelial cells in thecapillaries that supply the brain and central nervous system. Thisbarrier selectively allows entry of substances such as water, oxygen,carbon dioxide, and nonionic solutes such as glucose, alcohol, andgeneral anesthetics, while blocking entry of other substances. Somesmall molecules, such as amino acids, are taken across the barrier byspecific transport mechanisms. In one example, fenoterol or disclosedfenoterol analogues are capable of passing through the barrier.

Body Mass Index (BMI): A mathematical formula for measuring body mass inhumans, also sometimes called Quetelet's Index. BMI is calculated bydividing weight (in kg) by height² (in meters²). The current standardsfor both men and women accepted as “normal” are a BMI of 20-24.9 kg/m².In one embodiment, a BMI of greater than 25 kg/m² can be used toidentify an obese subject. Grade I obesity corresponds to a BMI of25-29.9 kg/m². Grade II obesity corresponds to a BMI of 30-40 kg/m²; andGrade III obesity corresponds to a BMI greater than 40 kg/m² (Jequier,Am. J Clin. Nutr., 45:1035-47, 1987). Ideal body weight will vary amongindividuals based on height, body build, bone structure, and sex.

Cannabinoid Receptors: A class of cell membrane receptors under the Gprotein-coupled receptor superfamily. The cannabinoid receptors containseven transmembrane spanning domains. Cannabinoid receptors areactivated by three major groups of ligands, endocannabinoids (producedby the mammalian body), plant cannabinoids (such as THC, produced by thecannabis plant) and synthetic cannabinoids (such as HU-210). All of theendocannabinoids and plant cannabinoids are lipophilic, i.e. fatsoluble, compounds. Two subtypes of cannabinoid receptors are CB₁ (seeGenBank Accession No. NM_033181 mRNA and UniProt P21554, each of whichis hereby incorporated by reference as of May 23, 2012) and CB2 (seeGenBank Accession No. NM 001841 mRNA and UniProt P34972, each of whichis hereby incorporated by reference as of May 23, 2012). The CB₁receptor is expressed mainly in the brain (central nervous system, CNS),but also in the lungs, liver and kidneys. The CB₂ receptor is expressedmainly in the immune system and in hematopoietic cells. Additionalnon-CB₁ and non-CB₂ include GPR55 (GenBank Accession No. NM_005683.3 orNP_005674.2 protein, each of which is hereby incorporated by referenceas of May 23, 2012), GPR119 (GenBank Accession No. NM 178471.2 orNP_848566.1 protein, each of which is hereby incorporated by referenceas of May 23, 2012) and GPR18 (also known as N-arachidonyl glycinereceptor and involved in microglial migration, GenBank Accession No.NM_001098200 mRNA, NP_001091670.1, each of which is hereby incorporatedby reference as of May 23, 2012).

The protein sequences of CB₁ and CB₂ receptors are about 44% similar.When only the transmembrane regions of the receptors are considered,amino acid similarity between the two receptor subtypes is approximately68%. In addition, minor variations in each receptor have beenidentified. Cannabinoids bind reversibly and stereo-selectively to thecannabinoid receptors. The affinity of an individual cannabinoid to eachreceptor determines the effect of that cannabinoid. Cannabinoids thatbind more selectively to certain receptors are more desirable formedical usage. GPR55 is coupled to the G-protein G₁₃ and/or G₁₁ andactivation of the receptor leads to stimulation of rhoA, cdc42 and rac1.GPR55 is activated by the plant cannabinoids Δ⁹-THC and cannabidiol, andthe endocannabinoids anandamide, 2-AG, noladin ether in the lownanomolar range. In contrast, CB1 and CB2 receptors are coupled toinhibitory G proteins. This indicates that both types of receptors willhave different readouts. For example, activation of CB1 causes apoptosiswhereas increase in GPR55 activity is oncogenic. The CB1 receptorantagonist (also termed ‘inverse agonist’) compound, AM251, is, in fact,an agonist for GPR55. It binds GPR55 and is readily internalized. Thisillustrates the opposite behavior of these two GPCRs. In turn, MNF isshown herein to be a CB1 receptor agonist (similar to WIN55,212-2) butacts as an inhibitor of GPR55, hence the pro-apoptotic behavior of MNFin select cancer cells.

As disclosed herein, specific fenoterol analogues, such as MNF and NF,are cannabinoid receptor regulators, such as regulators of GPR55. In anexample, a fenoterol analogue either alone or in combination with otheragents is administered to a subject to reduce or inhibit one or moresymptoms or signs associated with a disorder (such as a metabolic,inflammatory, pain or the like disorder) or disease (such ashepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, coloncancer, brain cancer, diabetes, or an inflammatory disease) modulated bycannabinoid receptors (such as GPR55).

Carbamate: A group of the formula —OC(O)N(R)—, wherein R is H, or analiphatic group, such as a lower alkyl group or an aralkyl group.

Chemotherapy; chemotherapeutic agents: As used herein, any chemicalagent with therapeutic usefulness in the treatment of diseasescharacterized by abnormal cell growth. Such diseases include tumors,neoplasms, and cancer as well as diseases characterized by hyperplasticgrowth. In one embodiment, a chemotherapeutic agent is an agent of usein treating neoplasms such as solid tumors, including a tumor associatedwith CB receptor activity and/or expression. In one embodiment, achemotherapeutic agent is radioactive molecule. In some embodiments, aCB receptor regulator, such as one or more fenoterol analogues or acombination thereof is a chemotherapeutic agent. In one example, achemotherapeutic agent is carmustine, lomustine, procarbazine,streptozocin, or a combination thereof. One of skill in the art canreadily identify a chemotherapeutic agent of use (e.g., see Slapak andKufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principlesof Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17in Abeloff, Clinical Oncology 2nd ed., © 2000 Churchill Livingstone,Inc; Baltzer L., Berkery R. (eds): Oncology Pocket Guide toChemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S,Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed.St. Louis, Mosby-Year Book, 1993).

Control or Reference Value: A “control” refers to a sample or standardused for comparison with a test sample. In some embodiments, the controlis a sample obtained from a healthy subject or a tissue sample obtainedfrom a patient diagnosed with a disorder or disease, such as a tumor,that did not respond to treatment with a β2-agonist. In someembodiments, the control is a historical control or standard referencevalue or range of values (such as a previously tested control sample,such as a group of subjects which do not have a tumor expressing CBreceptors or group of samples that represent baseline or normal values,such as the level of CB receptors in tumor tissue that does not respondto treatment with fenoterol, a fenoterol analogue or a combinationthereof).

Diabetes mellitus: A disease caused by a relative or absolute lack ofinsulin leading to uncontrolled carbohydrate metabolism, commonlysimplified to “diabetes,” though diabetes mellitus should not beconfused with diabetes insipidus. As used herein, “diabetes” refers todiabetes mellitus, unless otherwise indicated. A “diabetic condition”includes pre-diabetes and diabetes. Type 1 diabetes (sometimes referredto as “insulin-dependent diabetes” or “juvenile-onset diabetes”) is anautoimmune disease characterized by destruction of the pancreatic βcells that leads to a total or near total lack of insulin. In diabetestype 2 (sometimes referred to as “non-insulin-dependent diabetes” or“adult-onset diabetes”), the body does not respond to insulin, though itis present.

Symptoms of diabetes include: excessive thirst (polydipsia); frequenturination (polyuria); extreme hunger or constant eating (polyphagia);unexplained weight loss; presence of glucose in the urine (glycosuria);tiredness or fatigue; changes in vision; numbness or tingling in theextremities (hands, feet); slow-healing wounds or sores; and abnormallyhigh frequency of infection. Diabetes may be clinically diagnosed by afasting plasma glucose (FPG) concentration of greater than or equal to7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greaterthan or equal to 11.1 mmol/L (200 mg/dL) at about two hours after anoral glucose tolerance test (OGTT) with a 75 g load. A more detaileddescription of diabetes may be found in Cecil Textbook of Medicine, J.B. Wyngaarden, et al., eds. (W.B. Saunders Co., Philadelphia, 1992,19^(th) ed.).

A subject exhibiting one or more of the following risk factors isconsidered to have a heightened or substantial risk of developingdiabetes type 2:

1. Obesity, such as a BMI greater than or equal to about 30 kg/m²;

2. Elevated fasting blood glucose (FPG) levels;

3. Impaired glucose tolerance (IGT);

4. Non-Caucasian ethnicity;

5. Hyperinsulinemia;

6. Hypertriglyceridemia;

7. Family history of diabetes;

8. History of gestational diabetes;

9. Sedentary lifestyle; and

10. In humans, middle age or elderly status (i.e., 40 years old andolder).

A “non-diabetic” or “normal” subject does not have any form of diabetes,such as type 1 diabetes, type 2 diabetes, or pre-diabetes.

Derivative: A chemical substance that differs from another chemicalsubstance by one or more functional groups. Preferably, a derivative(such as a fenoterol analogue) retains a biological activity (CBreceptor activation) of a molecule from which it was derived (such as afenoterol analogue capable of regulating a CB receptor, such as GPR55).

Effective amount: An amount of agent that is sufficient to generate adesired response, such as reducing or inhibiting one or more signs orsymptoms associated with a condition or disease. When administered to asubject, a dosage will generally be used that will achieve target tissueconcentrations. In some examples, an “effective amount” is one thattreats one or more symptoms and/or underlying causes of any of adisorder or disease. In some examples, an “effective amount” is a“therapeutically effective amount” in which the agent alone with anadditional therapeutic agent(s) (for example a chemotherapeutic agent)induces the desired response such as treatment of a tumor. In oneexample, a desired response is to decrease tumor size or metastasis in asubject to whom the therapy is administered. Tumor metastasis does notneed to be completely eliminated for the composition to be effective.For example, a composition can decrease metastasis by a desired amount,for example by at least 20%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 98%, or even at least100% (elimination of the tumor), as compared to metastasis in theabsence of the composition.

In particular examples, it is an amount of an agent effective todecrease a number of carcinoma cells, such as in a subject to whom it isadministered, for example a subject having one or more carcinomas. Thecancer cells do not need to be completely eliminated for the compositionto be effective. For example, a composition can decrease the number ofcancer cells by a desired amount, for example by at least 20%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or even at least 100% (elimination of detectablecancer cells), as compared to the number of cancer cells in the absenceof the composition.

In some examples, an effective amount is the amount of (R,R′)- or(R,S′)-fenoterol analogue(s) useful in reducing, inhibiting, and/ortreating a disorder or disease associated with CB receptor, such asGPR55, expression and/or activity. Ideally, a therapeutically effectiveamount of an agent is an amount sufficient to reduce, inhibit, and/ortreat the disorder in a subject without causing a substantial cytotoxiceffect in the subject.

The effective amount of a composition useful for reducing, inhibiting,and/or treating a disorder in a subject will be dependent on the subjectbeing treated, the severity of the disorder, and the manner ofadministration of the therapeutic composition. Effective amounts atherapeutic agent can be determined in many different ways, such asassaying for a reduction in tumor size or improvement of physiologicalcondition of a subject having a tumor, such as a brain tumor. Effectiveamounts also can be determined through various in vitro, in vivo or insitu assays.

Glioblastoma: A common and malignant form of a primary brain tumor. Aglioblastoma is a grade IV astrocytoma and usually spreads rapidly inthe brain. In one example, a glioblastoma is inhibited by administeringa therapeutic effective amount of a fenoterol analogue with other agentscapable of regulating a CB receptor, such as GPR55, to a subject,thereby inhibiting one or more symptoms associated with theglioblastoma.

Inflammation: When damage to tissue occurs, the body's response to thedamage is usually inflammation. The damage may be due to trauma, lack ofblood supply, hemorrhage, autoimmune attack, transplanted exogenoustissue or infection. This generalized response by the body includes therelease of many components of the immune system (for instance, IL-1 andTNF), attraction of cells to the site of the damage, swelling of tissuedue to the release of fluid and other processes. In some examples, adisclosed fenoterol analogue capable of regulating CB receptor activityis used to treat, such as reduce or inhibit, one or more signs orsymptoms associated with inflammation.

Isomers: Compounds that have the same molecular formula but differ inthe nature or sequence of bonding of their atoms or the arrangement oftheir atoms in space are termed “isomers”. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers”.Stereoisomers that contain two or more chiral centers and are not mirrorimages of one another are termed “diastereomers.” Steroisomers that arenon-superimposable mirror images of each other are termed “enantiomers.”When a compound has an asymmetric center, for example, if a carbon atomis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCalm and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−) isomers, respectively). A chiralcompound can exist as either an individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds described herein may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R),(S), (R,R′), (R,S′)-stereoisomers or as mixtures thereof. Unlessindicated otherwise, the description or naming of a particular compoundin the specification and claims is intended to include both individualenantiomers and mixtures, racemic or otherwise, thereof. The methods forthe determination of stereochemistry and the separation of stereoisomersare well known in the art (see, e.g., March, Advanced Organic Chemistry,4th edition, New York: John Wiley and Sons, 1992, Chapter 4).

Obesity: A condition in which excess body fat may put a person at healthrisk (see Barlow and Dietz, Pediatrics 102: E29, 1998; NationalInstitutes of Health, National Heart, Lung, and Blood Institute (NHLBI),Obes. Res. 6 (suppl. 2):515-2095, 1998). Excess body fat is a result ofan imbalance of energy intake and energy expenditure. In one embodimentin humans, the Body Mass Index (BMI) is used to assess obesity. In oneembodiment, a BMI of 25.0 kg/m² to 29.9 kg/m² is overweight, while a BMIof 30 kg/m² is obese.

In another embodiment in humans, waist circumference is used to assessobesity. In this embodiment, in men a waist circumference of 102 cm ormore is considered obese, while in women a waist circumference of 89 cmor more is considered obese. Strong evidence shows that obesity affectsboth the morbidity and mortality of individuals. For example, an obeseindividual is at increased risk for heart disease, non-insulin-dependent(type 2) diabetes, hypertension, stroke, cancer (e.g., endometrial,breast, prostate, and colon cancer), dyslipidemia, gall bladder disease,sleep apnea, reduced fertility, and osteoarthritis, amongst others (seeLyznicki et al., Am. Fam. Phys. 63:2185, 2001).

Optional: “Optional” or “optionally” means that the subsequentlydescribed event or circumstance can but need not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not.

Oral glucose tolerance test (OGTT): A diagnostic test for diabetes.After fasting overnight, a subject is provided a concentrated sugarsolution to drink, usually containing 50 to 100 grams of glucose. Thesubject's blood is sampled periodically over the next few to severalhours to test blood glucose levels over time. In a non-diabetic subject,blood glucose concentration shows a slight upward shift and returns tonormal within 2-3 hours. In a diabetic subject, blood glucoseconcentration is generally higher than normal after fasting, rises moreafter the subject drinks the glucose solution, and may take severalhours to return to normal. An OGTT of greater than or equal to 140 mg/dland less than 200 mg/dl indicates that a subject has pre-diabetes. AnOGTT of greater than or equal to 200 mg/dl indicates that a subject hasfrank diabetes, and an OGTT of less than 140 mg/dl indicates that asubject is normal (healthy) and does not have pre-diabetes or diabetes.

Overweight: An individual who weighs more than their ideal body weight.An overweight individual can be obese, but is not necessarily obese. Inone embodiment, an overweight human individual is any individual whodesires to decrease their weight. In another embodiment, an overweighthuman individual is an individual with a BMI of 25.0 kg/m² to 29.9kg/m².

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more nucleic acidmolecules, proteins or antibodies that bind these proteins, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Phenyl: Phenyl groups may be unsubstituted or substituted with one, twoor three substituents, with substituent(s) independently selected fromalkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, halo,haloalkyl (such as —CF₃), nitro, cyano, —OR (where R is hydrogen oralkyl), —N(R)R′ (where R and R′ are independently of each other hydrogenor alkyl), —COOR (where R is hydrogen or alkyl) or —C(O)N(R′)R″ (whereR′ and R″ are independently selected from hydrogen or alkyl).

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpreparation is one in which a desired component such as an(R,R′)-enantiomer of fenoterol is more enriched than it was in apreceding environment such as in a (+)-fenoterol mixture. A desiredcomponent such as (R,R′)-enantiomer of fenoterol is considered to bepurified, for example, when at least about 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% of a sample by weight is composed of the desiredcomponent. Purity of a compound may be determined, for example, by highperformance liquid chromatography (HPLC) or other conventional methods.In an example, the specific fenoterol analogue enantiomers are purifiedto represent greater than 90%, often greater than 95% of the otherenantiomers present in a purified preparation. In other cases, thepurified preparation may be essentially homogeneous, wherein otherstereoisomers are less than 1%.

Compounds described herein may be obtained in a purified form orpurified by any of the means known in the art, including silica geland/or alumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. by Snyder and Kirkland, New York: JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed. by Stahl, NewYork: Springer Verlag, 1969. In an example, a compound includes purifiedfenoterol or fenoterol analogue with a purity of at least about 70%,80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weight relativeto other contaminants. In a further example, a compound includes atleast two purified stereoisomers each with a purity of at least about70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weightrelative to other contaminants. For instance, a compound can include asubstantially purified (R,R′)-fenoterol analogue and a substantiallypurified (R,S′-fenoterol analogue.

Subject: The term “subject” includes both human and veterinary subjects,for example, humans, non-human primates, dogs, cats, horses, rats, mice,and cows. Similarly, the term mammal includes both human and non-humanmammals.

Tissue: A plurality of functionally related cells. A tissue can be asuspension, a semi-solid, or solid. Tissue includes cells collected froma subject such as the brain or a portion thereof.

Tumor: All neoplastic cell growth and proliferation, whether malignantor benign, and all pre-cancerous and cancerous cells and tissues. Aprimary tumor is tumor growing at the anatomical site where tumorprogression began and proceeded to yield this mass. A primary braintumor (also referred to as a glioma) is a tumor that originates in thebrain. Exemplary primary brain tumors include astrocytomas,glioblastomas, ependymoma, oligodendroglomas, and mixed gliomas. In someexamples, a primary brain tumor expresses CB receptors, such as aglioblastoma associated with CB receptor expression.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. In one example, underconditions sufficient for includes administering one or more fenoterolanalogues, fenoterol or a combination thereof to a subject to at aconcentration sufficient to allow the desired activity. In someexamples, the desired activity is reducing or inhibiting a sign orsymptom associated with a disorder or disease, such as a primary braintumor, hepatocellular carcinoma, liver cancer, colon cancer, or lungcancer, can be evidenced, for example, by a delayed onset of clinicalsymptoms of the tumor in a susceptible subject, a reduction in severityof some or all clinical symptoms of the tumor, a slower progression ofthe tumor (for example by prolonging the life of a subject having thetumor), a reduction in the number of tumor reoccurrence, an improvementin the overall health or well-being of the subject, or by otherparameters well known in the art that are specific to the particulardisease. In one particulate example, the desired activity is preventingor inhibiting tumor growth, such as astrocytoma, glioblastoma, orhepatocellular carcinoma growth. Tumor growth does not need to becompletely inhibited for the treatment to be considered effective. Forexample, a partial reduction or slowing of growth such as at least abouta 10% reduction, such as at least 20%, at least about 30%, at leastabout 40%, at least about 50% or greater is considered to be effective.

III. (R,R′)-Fenoterol and Fenoterol Analogues

A. Chemical Structure

Some exemplary fenoterol analogues disclosed herein have the formula:

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl, aminocarbonyl or a combination thereof;

R₄ is H or lower alkyl;

R₅ is lower alkyl,

wherein X and Y independently are selected from hydrogen, lower —OR₆ and—NR₇R₈;

R₆ is lower alkyl or acyl; and

R₇ and R₈ independently are hydrogen, lower alkyl, alkoxy carbonyl, acylor amino carbonyl.

With continued reference to the general formula for fenoterol analoguesabove, Y may be —OH.

In one embodiment, R₅ is a 1- or 2-naphthyl derivative optionally having1, 2 or 3 substituents. Examples of such R₅ groups are represented bythe formula

wherein Y¹, Y² and Y³ independently are hydrogen, halogen,sulphur-containing moiety including SH, sulfoxides, sulphones,sulphanamides and related alkyl and aromatic substituted moieties, lower—OR₆ and —NR₇R₈; R₆ is independently for each occurrence selected fromlower alkyl and acyl; and R₇ and R₈ independently are hydrogen, loweralkyl, alkoxy carbonyl, acyl or amino carbonyl (carbamoyl). Inparticular compounds at least one of Y¹, Y² and Y³ is —OCH₃.

Particular R₅ groups include those represented by the formulas

wherein R₆ is lower alkyl, such as methyl, ethyl, propyl or isopropyl oracyl, such as acetyl.

Exemplary R₅ groups include

In one example, R₄ is lower alkyl and R₅ is

wherein X and Y independently are selected from H, lower alkyl —OR₆ and—NR₇R₈; R₆ is lower alkyl; and R₇ and R₈ independently are hydrogen orlower alkyl.

In a further example, R₄ is selected from ethyl, n-propyl, and isopropyland R₅ has the formula

wherein X is H, —OR₆ or —NR₇R₈. For example, R₆ may be methyl or R₇ andR₈ are hydrogen.

In an additional example, R₅ has the formula

In further embodiments, R₄ is selected from methyl, ethyl, n-propyl andisopropyl and R₅ represents

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a loweralkyl (such as, CH₃ or CH₂CH₃); R₅ is lower alkyl, or

wherein X, Y¹, Y² and Y³ independently are hydrogen, —OR₆ and —NR₇R₈; R₆is independently hydrogen, lower alkyl, acyl, alkoxy carbonyl or aminocarbonyl; R₇ and R₈ independently are hydrogen, lower alkyl, alkoxycarbonyl, acyl or amino carbonyl and wherein the compound is opticallyactive.

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a methyl oran ethyl; R₅ is

wherein X is —OH or —OCH₃.

In some embodiments, R₁-R₃ independently are hydrogen; R₄ is a methyl oran ethyl; R₅ is

Exemplary compounds include, but are not limited to,(R,R′)-4′-methoxy-1-naphthylfenoterol (MNF), (R,S′)-MNF,(R,R′)-ethylMNF, (R,R′)-naphthylfenoterol (NF), (R,R′)-ethylNF,(R,S′)-NF and (R,R′)-4′-amino-1-naphthylfenoterol (aminoNF), or(R,R′)-4′-hydroxy-1-naphthylfenoterol (hydroxyNF).

Examples of suitable groups for R₁-R₃ that can be cleaved in vivo toprovide a hydroxy group include, without limitation, acyl, acyloxy andalkoxy carbonyl groups. Compounds having such cleavable groups arereferred to as “prodrugs.” The term “prodrug,” as used herein, means acompound that includes a substituent that is convertible in vivo (e.g.,by hydrolysis) to a hydroxyl group. Various forms of prodrugs are knownin the art, for example, as discussed in Bundgaard, (ed.), Design ofProdrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology,Vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed), Designand Application of Prodrugs, Textbook of Drug Design and Development,Chapter 5, 113 191 (1991); Bundgaard, et al., Journal of Drug DeliveryReviews, 8:1 38 (1992); Bundgaard, Pharmaceutical Sciences, 77:285 etseq. (1988); and Higuchi and Stella (eds.) Prodrugs as Novel DrugDelivery Systems, American Chemical Society (1975).

In some embodiments, administering comprises administering atherapeutically effective amount of MNF, NF or a combination thereof. Insome embodiments, administering comprises administering atherapeutically effective amount of MNF.

In some embodiments, the method includes administering a therapeuticallyeffective amount of a pharmaceutical composition containing any of thedisclosed fenoterol analogues capable of regulating a CB receptordisorder or disease and a pharmaceutically acceptable carrier to treatthe disorder or disease regulated by a CB receptor, such as aglioblastoma or hepatocellular carcinoma expressing GPR55. For example,the disclosed (R,R′)-MNF, (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-NF,(R,R′)-ethylNF, (R,S′)-NF and (R,R′)-aminoNF, (R,R′)-hydroxyNF, or acombination thereof

An exemplary (R,R′)-compound has the chemical structure of:

X and R₁-R₃ are as described above.

An additional exemplary (R,R′)-compound has the chemical structure:

An exemplary (R,S′)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

An additional exemplary (R,S′)-compound has the chemical structure:

An exemplary (S,R′)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

An exemplary (S,S′)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

Examples of chemical structures illustrating the various stereoisomersof fenoterol are provided below.

Particular method embodiments contemplate the use of solvates (such ashydrates), pharmaceutically acceptable salts and/or different physicalforms of (R,R′)-fenoterol or any of the fenoterol analogues hereindescribed.

1. Solvates, Salts and Physical Forms

“Solvate” means a physical association of a compound with one or moresolvent molecules. This physical association involves varying degrees ofionic and covalent bonding, including by way of example covalent adductsand hydrogen bonded solvates. In certain instances the solvate will becapable of isolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. “Solvate”encompasses both solution-phase and isolable solvates. Representativesolvates include ethanol-associated compound, methanol-associatedcompounds, and the like. “Hydrate” is a solvate wherein the solventmolecule(s) is/are H₂O.

The disclosed compounds also encompass salts including, if severalsalt-forming groups are present, mixed salts and/or internal salts. Thesalts are generally pharmaceutically acceptable salts that arenon-toxic. Salts may be of any type (both organic and inorganic), suchas fumarates, hydrobromides, hydrochlorides, sulfates and phosphates. Inan example, salts include non-metals (e.g., halogens) that form groupVII in the periodic table of elements. For example, compounds may beprovided as a hydrobromide salt.

Additional examples of salt-forming groups include, but are not limitedto, a carboxyl group, a phosphonic acid group or a boronic acid group,that can form salts with suitable bases. These salts can include, forexample, nontoxic metal cations, which are derived from metals of groupsIA, IB, IIA and IIB of the periodic table of the elements. In oneembodiment, alkali metal cations such as lithium, sodium or potassiumions, or alkaline earth metal cations such as magnesium or calcium ionscan be used. The salt can also be a zinc or an ammonium cation. The saltcan also be formed with suitable organic amines, such as unsubstitutedor hydroxyl-substituted mono-, di- or tri-alkylamines, in particularmono-, di- or tri-alkylamines, or with quaternary ammonium compounds,for example with N-methyl-N-ethylamine, diethylamine, triethylamine,mono-, bis- or tris-(2-hydroxy-lower alkyl)amines, such as mono-, bis-or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine ortris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-loweralkyl)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine, or N-methyl-D-glucamine, or quaternaryammonium compounds such as tetrabutylammonium salts.

Exemplary compounds disclosed herein possess at least one basic groupthat can form acid-base salts with inorganic acids. Examples of basicgroups include, but are not limited to, an amino group or imino group.Examples of inorganic acids that can form salts with such basic groupsinclude, but are not limited to, mineral acids such as hydrochloricacid, hydrobromic acid, sulfuric acid or phosphoric acid. Basic groupsalso can form salts with organic carboxylic acids, sulfonic acids, sulfoacids or phospho acids or N-substituted sulfamic acid, for exampleacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid, and, in addition,with amino acids, for example with α-amino acids, and also withmethanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonicacid, ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid. In a currently preferred embodiment,fenoterol is provided as a hydrobromide salt and exemplary fenoterolanalogues are provided as their fumarate salts.

Additional counterions for forming pharmaceutically acceptable salts arefound in Remington's Pharmaceutical Sciences, 19th Edition, MackPublishing Company, Easton, Pa., 1995. In one aspect, employing apharmaceutically acceptable salt may also serve to adjust the osmoticpressure of a composition.

In certain embodiments the compounds used in the method are provided arepolymorphous. As such, the compounds can be provided in two or morephysical forms, such as different crystal forms, crystalline, liquidcrystalline or non-crystalline (amorphous) forms.

2. Use for the Manufacture of a Medicament

Any of the above described compounds (e.g., (R,R′) and/or (R,S′)fenoterol analogues or a hydrate or pharmaceutically acceptable saltthereof) or combinations thereof are intended for use in the manufactureof a medicament for regulation of a CB receptor, such as GPR55, in asubject either at risk of developing or having a CB receptor-regulateddisorder (such as a metabolic, inflammatory, pain or the like disorder)or disease (such as hepatocellular carcinoma, glioblastoma, livercancer, lung cancer, colon cancer, brain cancer, diabetes, or aninflammatory disease) modulated by cannabinoid receptors (such asGPR55).

Formulations suitable for such medicaments, subjects who may benefitfrom same and other related features are described elsewhere herein.

B. Methods of Synthesis

The disclosed fenoterol analogues can be synthesized by any method knownin the art including those described in U.S. patent application Ser. No.12/376,945 filed Feb. 9, 2009, U.S. patent application Ser. No.13/333,866 filed Dec. 21, 2011 and WO 2011/112867 filed Mar. 10, 2011,each of which is hereby incorporated by reference in its entirety. Manygeneral references providing commonly known chemical synthetic schemesand conditions useful for synthesizing the disclosed compounds areavailable (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein may be purified by any of the means knownin the art, including chromatographic means, such as HPLC, preparativethin layer chromatography, flash column chromatography and ion exchangechromatography. Any suitable stationary phase can be used, includingnormal and reversed phases as well as ionic resins. Most typically thedisclosed compounds are purified via open column chromatography or prepchromatography.

Suitable exemplary syntheses of fenoterol analogues are provided below:

Scheme I: An exemplary synthesis of 4 stereoisomers of 1-6 including thecoupling of the epoxide formed from either (R)- or(S)-3′,5′-dibenzyloxyphenylbromohydrin with the (R)- or (S)-enantiomerof the appropriate benzyl-protected 2-amino-3-benzylpropane (1-5) or the(R) or (S)-enantiomer of N-benzyl-2-aminoheptane (6).

Scheme II: Exemplary synthesis of (R)-7 and (S)-7 using2-phenethylamine. The resulting compounds may be deprotected byhydrogenation over Pd/C and purified as the fumarate salts.

Scheme III describes an exemplary synthesis for the chiral buildingblocks used in Scheme II. The (R)- and(S)-3′,5′-dibenzyloxyphenyl-bromohydrin enantiomers were obtained by theenantiospecific reduction of 3,5-dibenzyloxy-α-bromoacetophenone usingboron-methyl sulfide complex (BH₃SCH₃) and either (1R,2S)- or(1S,2R)-cis-1-amino-2-indanol. The required (R)- and(S)-2-benzylaminopropanes were prepared by enantioselectivecrystallization of the rac-2-benzylaminopropanes using either (R)- or(S)-mandelic acid as the counter ion.

IV. Pharmaceutical Compositions

The disclosed fenoterol analogues can be useful, at least, for reducingor inhibiting one or more symptoms or signs associated with a disorder(such as a metabolic, inflammatory, pain or the like disorder) ordisease (such as hepatocellular carcinoma, glioblastoma, liver cancer,lung cancer, colon cancer, brain cancer, diabetes, or an inflammatorydisease) modulated by cannabinoid receptors (such as GPR55).Accordingly, pharmaceutical compositions comprising at least onedisclosed fenoterol analogue are also described herein.

Formulations for pharmaceutical compositions are well known in the art.For example, Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes exemplaryformulations (and components thereof) suitable for pharmaceuticaldelivery of (R,R′)-fenoterol and disclosed fenoterol analogues.Pharmaceutical compositions comprising at least one of these compoundscan be formulated for use in human or veterinary medicine. Particularformulations of a disclosed pharmaceutical composition may depend, forexample, on the mode of administration (e.g., oral or parenteral) and/oron the disorder to be treated (e.g., a tumor associated with CBreceptor, such as GPR55 receptor, activity or expression). In someembodiments, formulations include a pharmaceutically acceptable carrierin addition to at least one active ingredient, such as a fenoterolcompound.

Pharmaceutically acceptable carriers useful for the disclosed methodsand compositions are conventional in the art. The nature of apharmaceutical carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions such as powder, pill, tablet, or capsuleforms conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can optionally contain minor amounts ofnon-toxic auxiliary substances or excipients, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like;for example, sodium acetate or sorbitan monolaurate. Other non-limitingexcipients include, nonionic solubilizers, such as cremophor, orproteins, such as human serum albumin or plasma preparations.

The disclosed pharmaceutical compositions may be formulated as apharmaceutically acceptable salt. Pharmaceutically acceptable salts arenon-toxic salts of a free base form of a compound that possesses thedesired pharmacological activity of the free base. These salts may bederived from inorganic or organic acids. Non-limiting examples ofsuitable inorganic acids are hydrochloric acid, nitric acid, hydrobromicacid, sulfuric acid, hydriodic acid, and phosphoric acid. Non-limitingexamples of suitable organic acids are acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, methyl sulfonic acid,salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid,gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid,p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists ofother suitable pharmaceutically acceptable salts are found inRemington's Pharmaceutical Sciences, 19th Edition, Mack PublishingCompany, Easton, Pa., 1995. A pharmaceutically acceptable salt may alsoserve to adjust the osmotic pressure of the composition.

The dosage form of a disclosed pharmaceutical composition will bedetermined by the mode of administration chosen. For example, inaddition to injectable fluids, oral dosage forms may be employed. Oralformulations may be liquid such as syrups, solutions or suspensions orsolid such as powders, pills, tablets, or capsules. Methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthe art.

Certain embodiments of the pharmaceutical compositions comprising adisclosed compound may be formulated in unit dosage form suitable forindividual administration of precise dosages. The amount of activeingredient such as (R,R′)-MNF or NF administered will depend on thesubject being treated, the severity of the disorder, and the manner ofadministration, and is known to those skilled in the art. Within thesebounds, the formulation to be administered will contain a quantity ofthe extracts or compounds disclosed herein in an amount effective toachieve the desired effect in the subject being treated.

In particular examples, for oral administration the compositions areprovided in the form of a tablet containing from about 1.0 to about 50mg of the active ingredient, particularly about 2.0 mg, about 2.5 mg, 5mg, about 10 mg, or about 50 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg (such as about 2 mg to about 10 mg) active ingredient isadministered two to four times a day, such as two times, three times orfour times.

In other examples, a suitable dose for parental administration is about1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose ofabout 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg,about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administeredparenterally. However, other higher or lower dosages also could be used,such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 toabout 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.

Single or multiple administrations of the composition comprising one ormore of the disclosed compositions can be carried out with dose levelsand pattern being selected by the treating physician. Generally,multiple doses are administered. In a particular example, thecomposition is administered parenterally once per day. However, thecomposition can be administered twice per day, three times per day, fourtimes per day, six times per day, every other day, twice a week, weekly,or monthly. Treatment will typically continue for at least a month, moreoften for two or three months, sometimes for six months or a year, andmay even continue indefinitely, i.e., chronically. Repeat courses oftreatment are also possible.

In one embodiment, the pharmaceutical composition is administeredwithout concurrent administration of a second agent for the treatment ofa tumor that expresses a CB receptor, such as GPR55. In one specific,non-limiting example, one or more of the disclosed compositions isadministered without concurrent administration of other agents, such aswithout concurrent administration of an additional agent also known totarget the tumor. In other specific non-limiting examples, atherapeutically effective amount of a disclosed pharmaceuticalcomposition is administered concurrently with an additional agent,including an additional therapy (such as, but not limited to, achemotherapeutic agent, an additional CB receptor regulator (such asregulator of GPR55), an anti-inflammatory agent, an anti-oxidant, orother agents known to those of skill in the art). For example, thedisclosed compounds are administered in combination with achemotherapeutic agent, anti-oxidants, anti-inflammatory drugs orcombinations thereof.

In other examples, a disclosed pharmaceutical composition isadministered an adjuvant therapy. For example, a pharmaceuticalcomposition containing one or more of the disclosed compounds isadministered orally daily to a subject in order to prevent or retardtumor growth. In one particular example, a composition containing equalportions of two or more disclosed compounds is provided to a subject. Inone example, a composition containing unequal portions of two or moredisclosed compounds is provided to the subject. For example, acomposition contains unequal portions of a (R,R′)-fenoterol derivativeand a (S,R′)-fenoterol derivative and/or a (R,S′)-derivative. In oneparticular example, the composition includes a greater amount of the(S,R′)- or (R,S′)-fenoterol derivative. Such therapy can be given to asubject for an indefinite period of time to inhibit, prevent, or reducetumor reoccurrence.

V. Methods of Use

The present disclosure includes methods of treating disorders includingreducing or inhibiting one or more signs or symptoms associated with adisorder (such as a metabolic, inflammatory, pain or the like disorder)or disease (such as hepatocellular carcinoma, glioblastoma, livercancer, lung cancer, colon cancer, brain cancer, diabetes, or aninflammatory disease) modulated by cannabinoid receptors (such asGPR55). In some examples, methods include reducing or inhibiting one ormore signs or symptoms associated with a tumor (such as hepatocellularcarcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, braincancer, diabetes, or an inflammatory disease) modulated by cannabinoidreceptors (such as GPR55).

In some examples, the tumor is a primary tumor, such as a primary braintumor expressing or regulated by CB receptors, such as GPR55. In someexamples, the tumor is a glioblastoma or hepatocellular carcinomaexpressing CB receptors, such as GPR55. In some examples, the tumor is aglioblastoma or hepatocellular carcinoma expressing CB receptors, suchas GPR55, but not expressing β2-AR. In some examples, the tumor is aglioblastoma or hepatocellular carcinoma expressing both CB receptors,such as GPR55, and β2-AR. The fenoterol analogue and/or fenoterol, suchas (R,R′) fenoterol, itself is administered depending upon the tumorreceptor population. For example, a tumor expressing or regulated by aCB receptors, such as GPR55, is treated by administering one or moredisclosed fenoterol analogues possessing CB receptor modulatoryactivity, such as (R,R′)-4′-methoxy-1-naphthylfenoterol (MNF),(R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-naphthylfenoterol (NF),(R,R′)-ethylNF, (R,S′)-NF and (R,R′)-4′-amino-1-naphthylfenoterol(aminoNF), (R,R′)-4′-hydroxy-1-naphthylfenoterol (hydroxyNF), or acombination thereof. In some examples, a tumor expressing or regulatedby a CB receptor, such as GPR55, and β2-AR is treated by administeringone or more disclosed fenoterol analogues possessing CB receptorregulatory activity, such as (R,R′)-4′-methoxy-1-naphthylfenoterol(MNF), (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-naphthylfenoterol (NF),(R,R′)-ethylNF, (R,S′)-NF and (R,R′)-4′-amino-1-naphthylfenoterol(aminoNF), (R,R′)-4′-hydroxy-1-naphthylfenoterol (hydroxyNF), and one ormore fenoterol analogues or fenoterol itself having β2-AR stimulatoryactivity in combination.

Disclosed methods include administering fenoterol, such as(R,R′)-fenoterol, a disclosed fenoterol analogue or a combinationthereof (and, optionally, one or more other pharmaceutical agents)depending upon the receptor population of the tumor, to a subject in apharmaceutically acceptable carrier and in an amount effective to treatthe tumor expressing a β2-AR, a CB receptor or combination thereof, suchas a primary tumor. Treatment of a tumor includes preventing or reducingsigns or symptoms associated with the presence of such tumor (forexample, by reducing the size or volume of the tumor or a metastasisthereof). Such reduced growth can in some examples decrease or slowmetastasis of the tumor, or reduce the size or volume of the tumor by atleast 10%, at least 20%, at least 50%, or at least 75%, such as between10%-90%, 20%-80%, 30%-70%, 40%-60%, including a 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% reduction.In another example, treatment includes reducing the invasive activity ofthe tumor in the subject, for example by reducing the ability of thetumor to metastasize. In some examples, treatment using the methodsdisclosed herein prolongs the time of survival of the subject.

Routes of administration useful in the disclosed methods include but arenot limited to oral and parenteral routes, such as intravenous (IV),intraperitoneal (IP), rectal, topical, ophthalmic, nasal, andtransdermal as described in detail above.

An effective amount of fenoterol, such as (R,R′)-fenoterol, or adisclosed fenoterol analogue or combination thereof will depend, atleast, on the particular method of use, the subject being treated, theseverity of the tumor, and the manner of administration of thetherapeutic composition. A “therapeutically effective amount” of acomposition is a quantity of a specified compound sufficient to achievea desired effect in a subject being treated. For example, this may bethe amount of (R,R′)-fenoterol, a disclosed fenoterol analogue or acombination thereof necessary to prevent or inhibit tumor growth and/orone or more symptoms associated with the tumor in a subject. Ideally, atherapeutically effective amount of (R,′R)-fenoterol or a disclosedfenoterol analogue is an amount sufficient to prevent or inhibit atumor, such as a brain or liver tumor growth and/or one or more symptomsassociated with the tumor in a subject without causing a substantialcytotoxic effect on host cells.

Therapeutically effective doses of a disclosed fenoterol compound orpharmaceutical composition can be determined by one of skill in the art,with a goal of achieving concentrations that are at least as high as theIC₅₀ of the applicable compound disclosed in the examples herein. Anexample of a dosage range is from about 0.001 to about 10 mg/kg bodyweight orally in single or divided doses. In particular examples, adosage range is from about 0.005 to about 5 mg/kg body weight orally insingle or divided doses (assuming an average body weight ofapproximately 70 kg; values adjusted accordingly for persons weighingmore or less than average). For oral administration, the compositionsare, for example, provided in the form of a tablet containing from about1.0 to about 50 mg of the active ingredient, particularly about 2.5 mg,about 5 mg, about 10 mg, or about 50 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg active ingredient is administered two to four times aday, such as two times, three times or four times.

In other examples, a suitable dose for parental administration is about1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose ofabout 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg,about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administeredparenterally. However, other higher or lower dosages also could be used,such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 toabout 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.

Single or multiple administrations of the composition comprising one ormore of the disclosed compositions can be carried out with dose levelsand pattern being selected by the treating physician. Generally,multiple doses are administered. In a particular example, thecomposition is administered parenterally once per day. However, thecomposition can be administered twice per day, three times per day, fourtimes per day, six times per day, every other day, twice a week, weekly,or monthly. Treatment will typically continue for at least a month, moreoften for two or three months, sometimes for six months or a year, andmay even continue indefinitely, i.e., chronically. Repeat courses oftreatment are also possible.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex and diet of the subject, mode and time of administration,rate of excretion, drug combination, and severity of the condition ofthe subject undergoing therapy.

Selecting a Subject

Subjects can be screened prior to initiating the disclosed therapies,for example to select a subject in need of or at risk of developing adisorder or disease regulated by CB receptor activity or expression.Briefly, the method can include screening subjects to determine if theyhave or are at risk of developing a GPR55-regulated disorder or disease,such as if the subject is in need of tumor inhibition. Subjects having atumor that expresses a CB receptor, such as GPR55, or is regulated by CBreceptor activity, such as a primary tumor, including a primary braintumor, such as a glioblastoma, hepatocellular carcinoma, liver cancer,lung cancer, or colon cancer or at risk of developing such a tumor areselected. In one example, subjects are diagnosed with the tumor byclinical signs, laboratory tests, or both. For example, a tumor, such asa primary brain tumor, can be diagnosed by characteristic clinicalsigns, such as headaches, vomiting, seizures, dizziness, weight loss andvarious associated complaints. Diagnosis is generally by imaginganalysis such as by magnetic resonance imaging (MRI) and confirmed byhistology. In some examples, a subject is selected that does not have ableeding disorder, such as an intracerebral hemorrhage.

In an example, a subject in need of the disclosed therapies is selectedby detecting a tumor expressing a CB receptor (e.g., GPR55) or regulatedby its activity, such as by detecting CB receptor activity or expressionin a sample obtained from a subject identified as having, suspected ofhaving or at risk of acquiring such a tumor. For example, detection ofaltered, such as at least a 10% alteration, including a 10%-90%,20%-80%, 30%-70%, 40%-60%, such as a 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% alteration or more inCB expression or activity as compared to CB expression or activity inthe absence of a primary tumor, indicates that the tumor can be treatedusing the fenoterol compositions and methods provided herein which areCB receptor regulators. In other examples, a subject is selected bydetecting a primary brain tumor such as an astrocytoma or glioblastomaby MRI or positron emission tomography (PET) in a subject.

In some examples, a subject is selected by determining the subject hasor is at risk of developing a disorder or disease, such as a tumorand/or cancer, which does not respond to β2-AR stimulation.

Pre-screening is not required prior to administration of the therapeuticagents disclosed herein (such as those including fenoterol, a fenoterolanalogue or a combination thereof).

Exemplary Tumors

Exemplary tumors include tumors that express a CB receptor, such asGPR55, or regulated by such, including primary tumors, such as a primarybrain tumor. A primary brain tumor includes astrocytomas, glioblastomas,ependymoma, oligodendroglomas, and mixed gliomas. Additional possibletypes of tumors associated with CB receptor activity or expressioninclude hematological tumors, such as leukemias, including acuteleukemias (such as 11q23-positive acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute myelogenous leukemia andmyeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (indolent and high grade forms), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of possible solid tumors which may express a CB receptor or beregulated by CB receptor activity, include sarcomas and carcinomas, suchas fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (including basal breast carcinoma,ductal carcinoma and lobular breast carcinoma), lung cancers, livercancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, medullary thyroid carcinoma, papillary thyroidcarcinoma, pheochromocytomas sebaceous gland carcinoma, papillarycarcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,seminoma, bladder carcinoma, and CNS tumors (such as a glioma,astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma and retinoblastoma). In several examples, atumor is a brain cancer, liver cancer, or lung cancer that expresses aCB receptor, such as GPR55. Tumors expressing a CB receptor, such asGPR55, can be identified by routine methods known to those of skill inthe art including Western blot and histological studies with antibodiescapable of detecting a CB receptor, such as GPR55

Assessment

Following the administration of one or more therapies, subjects having adisorder or disease regulated by CB receptor activity, such as atumor-expressing GPR55 (for example, a primary tumor) can be monitoredfor decreases in tumor growth, tumor volume or in one or more clinicalsymptoms associated with the tumor. In particular examples, subjects areanalyzed one or more times, starting 7 days following treatment.Subjects can be monitored using any method known in the art includingthose described herein including imaging analysis.

Additional Treatments and Additional Therapeutic Agents

In particular examples, if subjects are stable or have a minor, mixed orpartial response to treatment, they can be re-treated afterre-evaluation with the same schedule and preparation of agents that theypreviously received for the desired amount of time, including theduration of a subject's lifetime. A partial response is a reduction,such as at least a 10%, at least a 20%, at least a 30%, at least a 40%,at least a 50%, or at least a 70% reduction in one or more signs orsymptoms associated with the disorder or disease, such as a tumorregulated by CB receptor activity, including tumor size or volume.

In some examples, the method further includes administering atherapeutic effective amount of fenoterol, a fenoterol analogue or acombination thereof with additional therapeutic treatments. Inparticular examples, prior to, during, or following administration of atherapeutic amount of an agent that prevents or inhibits a tumorregulated by CB receptor activity, the subject can receive one or moreother therapies. In one example, the subject receives one or moretreatments to remove or reduce the tumor prior to administration of atherapeutic amount of a composition including fenoterol, a fenoterolanalogue or combination thereof.

Examples of such therapies include, but are not limited to, surgicaltreatment for removal or reduction of the tumor (such as surgicalresection, cryotherapy, or chemoembolization), as well as anti-tumorpharmaceutical treatments which can include radiotherapeutic agents,anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agentsand antioxidants, kinase inhibitors, and other agents. Particularexamples of additional therapeutic agents that can be used includemicrotubule-binding agents, DNA intercalators or cross-linkers, DNAsynthesis inhibitors, DNA and/or RNA transcription inhibitors,antibodies, enzymes, enzyme inhibitors, and gene regulators. Theseagents (which are administered at a therapeutically effective amount)and treatments can be used alone or in combination. Methods andtherapeutic dosages of such agents are known to those skilled in theart, and can be determined by a skilled clinician.

“Microtubule-binding agent” refers to an agent that interacts withtubulin to stabilize or destabilize microtubule formation therebyinhibiting cell division. Examples of microtubule-binding agents thatcan be used in conjunction with the disclosed therapy include, withoutlimitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine(navelbine), the epothilones, colchicine, dolastatin 15, nocodazole,podophyllotoxin and rhizoxin. Analogs and derivatives of such compoundsalso can be used and are known to those of ordinary skill in the art.For example, suitable epothilones and epothilone analogs are describedin International Publication No. WO 2004/018478. Taxoids, such aspaclitaxel and docetaxel, as well as the analogs of paclitaxel taught byU.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.

The following classes of compounds are of use in the methods disclosedherein: Suitable DNA and/or RNA transcription regulators, including,without limitation, actinomycin D, daunorubicin, doxorubicin andderivatives and analogs thereof also are suitable for use in combinationwith the disclosed therapies. DNA intercalators and cross-linking agentsthat can be administered to a subject include, without limitation,cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C,bleomycin, chlorambucil, cyclophosphamide and derivatives and analogsthereof. DNA synthesis inhibitors suitable for use as therapeutic agentsinclude, without limitation, methotrexate, 5-fluoro-5′-deoxyuridine,5-fluorouracil and analogs thereof. Examples of suitable enzymeinhibitors include, without limitation, camptothecin, etoposide,formestane, trichostatin and derivatives and analogs thereof. Examplesof alkylating agents include carmustine or lomustine. Suitable compoundsthat affect gene regulation include agents that result in increased ordecreased expression of one or more genes, such as raloxifene,5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen, 4-hydroxytamoxifen,mifepristone and derivatives and analogs thereof. Kinase inhibitorsinclude Gleevac, Iressa, and Tarceva that prevent phosphorylation andactivation of growth factors.

Other therapeutic agents, for example anti-tumor agents, that may or maynot fall under one or more of the classifications above, also aresuitable for administration in combination with the disclosed therapies.By way of example, such agents include adriamycin, apigenin, rapamycin,zebularine, cimetidine, and derivatives and analogues thereof.

In one example, at least a portion of the tumor (such as the primarybrain tumor) is surgically removed (for example via cryotherapy),irradiated, chemically treated (for example via chemoembolization) orcombinations thereof, prior to administration of the disclosed therapies(such as administration of fenoterol, a fenoterol analogue or acombination thereof). For example, a subject having a primary braintumor associated with CB receptor activity can have at least a portionof the tumor surgically excised prior to administration of the disclosedtherapies. In an example, one or more chemotherapeutic agents areadministered following treatment with a composition including fenoterol,a fenoterol analogue or a combination thereof. In another particularexample, the subject has a primary brain tumor and is administeredradiation therapy, chemoembolization therapy, or both concurrently withthe administration of the disclosed therapies.

Additional Disorders and Diseases

As discussed above, in addition to methods of treating CBreceptor-regulated tumors, it is contemplated that the disclosedfenoterol analogues possessing CB receptor modulatory activity, such asmodulator of GPR55 activity, can be used to treat other conditionsassociated with CB receptor regulation, such as metabolic disorders anddisease (e.g., obesity and diabetes), or inflammatory and neuropathicpain disorders, diseases associated with aging such as Alzheimer's, boneloss, muscle wasting (sarcopenia), osteoarthritis and loss of appetite,central nervous system conditions such as depression and anxiety andother diseases and disorders associated with CB receptor regulation.

Based on these observations, methods of treating metabolicdisorders/diseases, (such as obesity, loss of appetite, and/ordiabetes), bone and/or muscle disorders or diseases (such as bone loss,muscle wasting, osteoarthritis), central nervous system conditions (suchas depression and anxiety) and inflammatory disorders/diseases, such asinflammation, neuropathic pain disorders and diseases associated withinflammation and/or aging (such as Alzheimer's disease, osteoarthritis),are disclosed. For example, disclosed herein are methods of preventingand treating obesity, diabetes, and related disorders.

In one example, a method of treating obesity or a condition associatedwith obesity, such as diabetes, in a subject is disclosed comprisingadministering to a subject an effective amount of at least one fenoterolanalogue with CB receptor modulatory activity, such as GPR55 activity(e.g., a disclosed naphthylfenoterol analogue, such as(R,R′)-4′-methoxy-1-naphthylfenoterol (MNF), (R,S′)-MNF,(R,R′)-ethylMNF, (R,R′)-naphthylfenoterol (NF), (R,R′)-ethylNF,(R,S′)-NF and (R,R′)-4′-amino-1-naphthylfenoterol (aminoNF),(R,R′)-4′-hydroxy-1-naphthylfenoterol (hydroxyNF), or a combinationthereof), to reduce or inhibit one or more signs or symptoms associatedwith obesity or a condition associated with obesity. In some examples,methods of treating one or more signs or symptoms associated with aninflammatory disorder and/or disease are disclosed comprisingadministering to a subject an effective amount of at least one fenoterolanalogue with CB receptor modulatory activity, such as GPR55 activity(e.g., a disclosed naphthylfenoterol analogue, such as(R,R′)-4′-methoxy-1-naphthylfenoterol (MNF), (R,S′)-MNF,(R,R′)-ethylMNF, (R,R′)-naphthylfenoterol (NF), (R,R′)-ethylNF,(R,S′)-NF and (R,R′)-4′-amino-1-naphthylfenoterol (aminoNF),(R,R′)-4′-hydroxy-1-naphthylfenoterol (hydroxyNF), or a combinationthereof), to reduce or inhibit one or more signs or symptoms associatedwith the inflammatory disorder/disease or a condition associated withthe inflammatory disorder/disease.

Disclosed methods include administering a disclosed fenoterol analoguewith CB receptor modulatory activity (and, optionally, one or more otherpharmaceutical agents) to a subject in a pharmaceutically acceptablecarrier and in an amount effective to treat the disorder or diseaseregulated by CB receptor activity, such as GPR55 activity. Treatment ofthe disorder or disease includes preventing or reducing signs orsymptoms associated with the particular disorder or disease. The signsand symptoms associated with the particular disorder or disease areknown to one of ordinary skill in the art and can be measured by assaysdisclosed herein as well as those known to those skilled in the art. Insome examples, treatment using the methods disclosed herein prolongs thetime of survival of the subject.

Routes of administration useful in the disclosed methods include but arenot limited to oral and parenteral routes, such as intravenous (IV),intraperitoneal (IP), rectal, topical, ophthalmic, nasal, andtransdermal as described in detail above.

An effective amount of a disclosed fenoterol analogue or combinationthereof will depend, at least, on the particular method of use, thesubject being treated, the severity of the disorder/disease, and themanner of administration of the therapeutic composition. A“therapeutically effective amount” of a composition is a quantity of aspecified compound sufficient to achieve a desired effect in a subjectbeing treated. Ideally, a therapeutically effective amount of adisclosed fenoterol analogue is an amount sufficient to prevent orinhibit one or more symptoms associated with the particulardisorder/disease in a subject without causing a substantial cytotoxiceffect on host cells.

Therapeutically effective doses of a disclosed fenoterol compound orpharmaceutical composition can be determined by one of skill in the art,with a goal of achieving concentrations that are at least as high as theIC₅₀ of the applicable compound disclosed in the examples herein. Anexample of a dosage range is from about 0.001 to about 10 mg/kg bodyweight orally in single or divided doses. In particular examples, adosage range is from about 0.005 to about 5 mg/kg body weight orally insingle or divided doses (assuming an average body weight ofapproximately 70 kg; values adjusted accordingly for persons weighingmore or less than average). For oral administration, the compositionsare, for example, provided in the form of a tablet containing from about1.0 to about 50 mg of the active ingredient, particularly about 2.5 mg,about 5 mg, about 10 mg, or about 50 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg active ingredient is administered once to four times aday, such as one time, two times, three times or four times.

In other examples, a suitable dose for parental administration is about1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose ofabout 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg,about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administeredparenterally. However, other higher or lower dosages also could be used,such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 toabout 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.

Single or multiple administrations of the composition comprising one ormore of the disclosed compositions can be carried out with dose levelsand pattern being selected by the treating physician. Generally,multiple doses are administered. In a particular example, thecomposition is parenterally administered once per day. However, thecomposition can be administered twice per day, three times per day, fourtimes per day, six times per day, every other day, twice a week, weekly,or monthly. Treatment will typically continue for at least one month,more often for two or three months, sometimes for six months or a year,and may even continue indefinitely, that is, chronically. Repeat coursesof treatment are also possible.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex and diet of the subject, mode and time of administration,rate of excretion, drug combination, and severity of the condition ofthe subject undergoing therapy. In some examples, one or more disclosedfenoterol analogues with CB receptor activity is orally administered toa subject daily to treat one or more symptoms associated with an agingdisorder or disease (such as Alzheimer's, sarcopenia, bone loss, orcombinations thereof) or a central nervous system disorder or disease(such as anxiety or depression).

Subjects can be screened prior to initiating the disclosed therapies,for example to select a subject in need of or at risk of developing adisorder or disease regulated by CB receptor activity or expression.Briefly, the method can include screening subjects to determine if theyhave or are at risk of developing a GPR55-regulated disorder or disease.Subjects having a disorder or disease that expresses a CB receptor, suchas GPR55, or is regulated by CB receptor activity are selected. In oneexample, subjects are diagnosed by clinical signs, laboratory tests, orboth known to those of ordinary skill in the art or disclosed herein (orboth).

Pre-screening is not required prior to administration of the therapeuticagents disclosed herein (such as those including fenoterol, a fenoterolanalogue or a combination thereof).

In particular examples, if subjects are stable or have a minor, mixed orpartial response to treatment, they can be re-treated afterre-evaluation with the same schedule and preparation of agents that theypreviously received for the desired amount of time, including theduration of a subject's lifetime. A partial response is a reduction,such as at least a 10%, at least a 20%, at least a 30%, at least a 40%,at least a 50%, or at least a 70% reduction in one or more signs orsymptoms associated with the disorder or disease.

In some examples, the method further includes administering atherapeutic effective amount of one or more fenoterol analogues withadditional therapeutic treatments. In particular examples, prior to,during, or following administration of a therapeutic amount of an agentthat prevents or inhibits a tumor regulated by CB receptor activity, thesubject can receive one or more other therapies. In one example, thesubject receives one or more treatments to remove or reduce one or moresigns or symptoms associated with the CB receptor regulateddisorder/disease prior to administration of a therapeutic amount of acomposition including one or more fenoterol analogues.

In particular examples, prior to, during, or following administration ofa therapeutic amount of a disclosed fenoterol analogue composition thatreduces or inhibits one or more signs or symptoms of obesity or acondition associated with obesity, the subject can receive one or moreother therapies. In one example, the subject receives one or moretreatments to remove or reduce one or more conditions associated withobesity, such as diabetes.

Examples of such therapies include, but are not limited to,anti-diabetic agents, insulin sensitizers, insulin secretagogues, agentsthat preserve and/or increase β-cell mass, agents that enhanceglucose-stimulated insulin secretion and glucose uptake in peripheralorgans of insulin action (skeletal muscle, liver, adipose tissue),agents that suppress endogenous glucose production, and anti-obesityagents. For example, the disclosed therapies can be administered withanti-diabetic agents such as biguanides. In particular embodiments, thebiguanide antidiabetic agent is metformin. Metformin is manufactured byLyonnaise Industrielle Pharmaceutique SA (Lyons, France), also known byits acronym LIPHA SA, and commercially distributed in the United Statesas a hydrochloride salt by the Bristol-Myers Squibb Company (Princeton,N.J.) as GLUCOPHAGE® XR. Additionally, Bristol-Myers Squibb distributesa pharmaceutical having a combination of metformin and glyburide asGLUCOVANCE®.

Anti-diabetic agents other than biguanides can also be administered tothe identified subject. For example, in alternative embodiments, theanti-diabetic agent is a thiazolidinedione, such as troglitazone. Insome examples, the anti-diabetic agent is an incretin or dipeptidylpeptidase-4 inhibitor, but the anti-diabetic agent can be any agent ofinterest.

A therapeutically effective amount of an anti-diabetic agent may beadministered in a single dose, or in several doses, for example daily,during a course of treatment. The course of treatment may last for anylength of time, such as a day or several days, a week or several weeks,a month or several months, or a year or several years, so long as thetherapeutic effect is observed, such as inhibiting the onset of type IIdiabetes in a subject diagnosed with pre-diabetes, or inducing a subjectdiagnosed with type 2 diabetes or pre-diabetes to a normal glucosetolerance. The subject can be monitored while undergoing treatment usingthe methods described herein in order to assess the efficacy of thetreatment protocol. In this manner, the length of time or the amountgiven to the subject can be modified based on the results obtained usingthe methods disclosed herein.

The therapeutically effective amount will depend on the anti-diabeticagent being used, the characteristics of the subject being treated (suchas age, BMI, physiological condition, etc.), the severity and type ofthe affliction, and the manner of administration of the agent. Thetherapeutically effective dose can be determined by various methods,including generating an empirical dose-response curve, predictingpotency and efficacy by using quantitative structure-activityrelationships (QSAR) methods or molecular modeling, and other methodsused in the pharmaceutical sciences. In certain, non-limiting examples,the therapeutically effective amount of metformin (or a relatedbiguanide analog or homolog) is at least about 1000 mg per day, such asat least about 1500 mg per day, or even at least about 1700 mg per day.In certain other, non-limiting examples, the total amount of metforminis divided into smaller doses, such as two or three doses per day, forexample 850 mg twice a day (b.i.d.) or 500 mg three times a day(t.i.d.). In alternative, non-limiting examples, the total amount ofmetformin is about 500 mg or less per day. The subject can be monitoredat different doses of an agent using the assays described herein, inorder to determine a therapeutically effective amount for the subject ofinterest.

For administration to animals, purified therapeutically active agentsare generally combined with a pharmaceutically acceptable carrier.Pharmaceutical preparations may contain only one type of anti-diabeticagent or may be composed of a combination of several types ofanti-diabetic agents, such as a combination of two or more anti-diabeticagents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Anti-diabetic agents may be administered by any means that achieve theirintended purpose. For example, the anti-diabetic agents may beadministered to a subject through systemic administration, such asintravenous, intraperitoneal, intralesional, suppository, or oraladministration.

The anti-diabetic agent can be administered alone or in combination withanother anti-diabetic agent. In certain embodiments, the anti-diabeticagent is administered in the absence of administering any otheranti-diabetic agent.

Other measures may be taken to inhibit or delay the onset of type IIdiabetes in subjects at a heightened risk of developing the disease. Forexample, in some embodiments, a subject may be instructed, trained, orinduced to adopt anti-diabetic lifestyle modifications. For example, thesubject can be counseled to reduce caloric intake or to exercise. Themethods disclosed herein can be used to monitor the effectiveness ofthese alternative measures to determine if pharmaceutical interventionis warranted for a subject of interest.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLES Example 1 Material and Methods

This example describes the Material and Methods used for Examples 2-4.

Materials.

(R,R′)-, (R,S′)-, (S,R′)- and (S,S′)-fenoterol and the fenoterolanalogs, (R,R′)-ethylfenoterol, (R,R′)-4′-aminofenoterol,(R,R′)-1-naphthylfenoterol and (R,R′)- and(R,S′)-4-methoxy-1-naphthylfenoterol, were synthesized as previouslydescribed (Jozwiak et al., J Med Chem 50:2903-2915, 2007; Jozwiak etal., Bioorg Med Chem 18:728-736, 2010; each of which is incorporated byreference in its entirety). [³H]-Thymidine (70-90 Ci/mmol) was purchasedfrom PerkinElmer Life and Analytical Sciences (Waltham, Mass.). Eagle'sMinimum Essential Medium (E-MEM), trypsin solution, phosphate-bufferedsaline (PBS), fetal bovine serum (FBS), 100× solutions of sodiumpyruvate (100 mM), L-glutamine (200 mM), and penicillin/streptomycin (amixture of 10,000 units/ml penicillin and 10,000 μg/ml streptomycin)were obtained from Quality Biological (Gaithersburg, Md.). WIN 55,212-2,AM251, and AM630 were purchased from Cayman Chemical (Aim Arbor, Mich.).ICI 118,551 hydrochloride and (R)-isoproterenol were obtained fromSigma-Aldrich (St. Louis, Mo.).

Maintenance and Treatment of Cell Lines.

Human HepG2 hepatocarcinoma cells and human U87MG glioma cells (ATCC,Manassas, Va.) were maintained in EMEM medium supplemented with 1%L-glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin, and 10% FBS(Hyclone, Logan, Utah). The human 1321N1 astrocytoma cells (EuropeanCollection of Cell Cultures, Sigma-Aldrich) were cultured in Dulbecco'smodified Eagle's medium supplemented with 10% FBS andpenicillin/streptomycin. All cell lines were cultured at 37° C. in 5%CO₂, and the medium was replaced every 2-3 days.

Unless otherwise indicated, cells at 70-80% confluence were depleted ofserum for 3 hours, followed by the addition of ICI 118551, AM251, AM630or WIN 55-212,2 for 1 hour before treatment with vehicle, fenoterol orfenoterol derivatives at the indicated concentrations.

[³H]-Thymidine Incorporation Assay.

Cells were seeded in 12-well plates at approximately 50,000 cells/welland incubated at 37° C. After 24 hours, the wells were rinsed with PBSand replaced with serum-free medium containing the appropriateconcentration of the test compounds. After another 24-hour incubation at37° C., 1 μCi of [³H]-thymidine was added to each well and incubated at37° C. for 16 hours. [³H]-Thymidine incorporation into DNA was monitoredafter the cells were washed twice with PBS and then lysed in 600 μL of0.1 N NaOH for 30 minutes with shaking. The lysate was then mixed with 3mL of liquid scintillation cocktail (Beckman Coulter, Inc., Brea,Calif.), and radioactivity was measured by liquid scintillation countingusing Beckman Coulter LS6000IC Scintillation Counter. Data are shown asCPM incorporated compared to the control cells.

Camp Accumulation.

HepG2 cells were seeded in 96-well plates and grown to confluency. Cellswere rinsed in Krebs-HEPES buffer, pH 7.4, pre-incubated for 10 minuteswith the buffer, and then 10 μM (R)-isoproterenol or (R,R′)-fenoterolwas added followed by incubation for an additional 10 minutes. Thelevels of cAMP accumulated in cells were determined and normalized tothe amount of protein per well.

RNA Extraction, cDNA Synthesis, and RT-PCR Analysis.

Total RNA was isolated from HepG2, 1321N1 and U87MG cells using theRNeasy Mini kit (Qiagen, Valencia, Calif.). The RNA preparation includeda DNAse digestion step. RNA concentration and quality was measured usingthe NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington,Del.). To obtain cDNA, 1 μg total RNA was reverse-transcribed using thePromega reverse transcription kit (Promega Corp., Madison, Wis.). PCRreactions were performed to determine the expression of CB1R, CB2R, andβ2-AR mRNAs using GAPDH as internal control. The PCR primers andconditions are found in Supplemental Table 1.

Cell Cycle Analysis.

Cell cycle distributions were performed by flow cytometry on propidiumiodide-stained nuclei prepared by the NM technique. DNA histograms of atleast 10,000 cells acquired on a Becton-Dickinson FACScanto II (BDBiosciences, San Jose, Calif.) were deconvoluted using the Multicycleprogram (Phoenix Flow Systems) for estimates of the percentage of cellsin the G0/1, S, and G2+M phases of the cell cycle. Debris and doubletswere removed from the analysis by software algorithms.

Apoptosis Assay.

The degree of apoptosis induced by drug treatment was assayed by flowcytometry using the Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit(Invitrogen) following the standard manufacturer's protocol. Briefly,HepG2 cells (5×10⁵) were grown on 100-mm dishes for 24 hours followed bytreatment with vehicle, (R,R′)-fenoterol, or (R,R′)-MNF, all inserum-free medium. Cells were subsequently harvested after a 24-hourincubation, washed in cold PBS, and resuspended in 100 μL of 1×annexin-binding buffer to maintain a density ˜1×10⁶ cells/mL, afterwhich 5 μL Alexa Fluor® 488 annexin V and 1 μL 100 μg/mL propidiumiodide were added to the cell suspensions. Cells were then incubated atroom temperature for 15 minutes and 400 μL 1× annexin-binding buffer wasadded followed by gentle mixing. Stained cells were analyzed on aBDFACSCanto II flow cytometer.

Western Blotting.

Cells were lysed with RIPA buffer containing EGTA and EDTA (BostonBioProducts, Ashland, Mass.). The lysis buffer was mixed with a proteaseinhibitor cocktail (Sigma-Aldrich). Protein concentrations were measuredusing the bicinchoninic acid reagent (Thermo-Pierce Biotechnology, Inc.,Rockford, Ill.). Proteins (20 μg/well) were separated on 4-12% precastgels (Invitrogen, Carlsbad, Calif.) using SDS-polyacrylamide gelelectrophoresis under reducing conditions and were electrophoreticallytransferred onto polyvinylidene fluoride membrane (Invitrogen). Westernblots were performed according to standard methods. The visualization ofimmunoreactive bands was performed using the ECL Plus Western BlottingDetection System (GE Healthcare, N.J.) and their quantification was doneby volume densitometry using Image J software and normalization toβ-actin. The primary antibody for β2-AR was obtained from Enzo LifeSciences, Inc. (Cat. # ADI-905-742-100, Farmingdale, N.Y.); rabbitanti-phospho-Akt (Ser-473), phospho-ERK1/2, total Akt and total ERK2were from Cell Signaling Technology (Beverly, Mass.), and anti-β-actinwas from Abcam (Cambridge, Mass.). The antibodies were used at adilution recommended by the manufacturer.

Statistical Analysis.

Results were expressed as relative to the control value. Studies wereperformed in at least two to three different culture preparations, andtwo to three dishes for each test condition were plated in eachpreparation. Results are expressed as means±S.E. Student's t-test wasused to make statistical comparisons between groups. Analyses wereperformed using the SigmaPlot Software (Systat Software, Inc. San Jose,Calif.), Graphpad Prism 4 (GraphPad Software, Inc., La Jolla, Calif.)and Microsoft® Office Excel, 2003 (Microsoft Corp., Redmond, Wash.),with p values ≤0.05 considered significant.

Example 2 Characterization of Fenoterol and Fenoterol Analogs onCannabinoid Receptor Activity

This example describes a series of studies performed to characterize theability of fenoterol and disclosed fenoterol analogs to modulatecannabinoid receptor activity.

(R,R′)-Fenoterol, (R,R′)-Fen, is a potent and selective agonist of theβ2-adrenergic receptor (β2-AR), which has a 43-fold higher affinity forthe β2-AR relative to the β₁-AR and an EC_(50cAMP) value of 0.3 nM forthe stimulation of cAMP accumulation in HEK cells expressing humanβ2-AR. The inventors have disclosed the synthesis and characterizationof a number of (R,R′)-Fen analogs and stereoisomers with a range ofβ₂-AR selectivity and potency (see, for example International PatentPublication No. WO 2008/022038 and WO 2011/112867, each of which isincorporated by reference in its entirety herein). One of these analogs,(R,R′)-4-methoxy-1-naphthylfenoterol (MNF) has a β2-AR selectivity of573 with an EC_(50cAMP) of 3.90 nM.

β₂-ARs associate with heterotrimeric G proteins (e.g., G_(s), G_(i)),ion channels and cytosolic scaffold proteins, including β-arrestin, toinitiate various signaling pathways and modulate the activity ofintracellular effectors such as adenyl cyclase and mitogen-activatedprotein kinase (MAPK). The difference in the G protein and β-arrestinsignaling by β₂-AR agonists has been attributed to interaction withligand-specific GPCR conformations and functional selectivity, which isbased upon the assumption that the β₂-AR exists in an inactive (R) stateand one or more ligand-specific active conformations (R*^(n)). The basisfor the ligand-specific differences in pharmacological outcome lies inthe interplay between the molecular structure of the agonist and thecellular environment of the receptor. In the first instance, theinventors have shown that the G_(s)/G_(i) selectivity of Fen is afunction of molecular structure and stereochemistry as (R,R′)-Fenpreferentially activated G_(s) signaling in a cardiomyocytecontractility model while (S,R′)-Fen and (R,R′)-MNF activated both G_(s)and G_(i) proteins. In the latter case, the inventors have demonstratedthat β2-AR agonists such as (R,R′)-Fen and isoproterenol exertanti-proliferative effects in the human-derived 1321N1 astrocytoma cellline specifically through the cAMP-dependent pathway while Yuan andcolleagues (Oncol Rep 23:151-157, 2010) reported that isoproterenoldose-dependently induced the growth of the human-derived HepG2hepatocellular carcinoma cell line.

The current example describes the effect of the molecular structure andstereochemistry of β₂-AR agonists on [³H]-thymidine incorporation inHepG2 cells and to compare these results to similar studies conductedusing the 1321N1 and human-derived U87MG glioblastoma cells lines. Theinitial data demonstrated that (R,R′)-Fen and isoproterenol induced[³H]-thymidine incorporation in HepG2 cells, reduced proliferation in1321N1 cells and had no effect on U87MG cells and that the effects inthe HepG2 and 1321N1 cells could be attenuated by the β₂-AR antagonistICI 118551. When (R,R′)-MNF was utilized in the studies, oppositeresults were obtained as the compound inhibited [³H]-thymidineincorporation in HepG2 cells and had no significant effect on 1321N1cells. The inhibitory effect of (R,R′)-MNF in HepG2 cells was notaffected by the addition of ICI 118551 and (R,R′)-MNF also attenuated[³H]-thymidine incorporation in the β2-AR-deficient U87MG cell line.These results indicate that while (R,R′)-MNF is a full β2-AR agonist,the anti-proliferative effects of this compound are not due to thisactivity. Further studies indicated that AM251 and AM630, inverseagonists of the CB1 and CB2 cannabinoid receptors, respectively, blocked(R,R′)-MNF mitogenic responses in HepG2 and U87MG cell lines and thatWIN 55,212-2, a synthetic CB1 and CB2 receptor agonist, produced growthinhibition in these cell lines. These results suggest that cannabinoidreceptor activation is associated with the cell type-dependentantiproliferative and pro-apoptotic effects of (R,R′)-MNF.

Expression of β2-AR in Select Human Cancer Cell Lines.

The protein levels of the β2-AR were determined by Western blot analysisin total extracts of HepG2 hepatocarcinoma cells, 1321N1 astrocytomacells, and U87MG glioma cells (FIG. 1A). β2-AR protein level was thehighest in 1321N1 cells when compared to HepG2 cells. U87MG cells werepreviously found by the inventors to be devoid of β₂-AR at the cellsurface.

Effect of β-AR Agonists on cAMP Accumulation and Phosphorylation of Aktand ERK1/2 in HepG2 Cells.

Neither (R)-isoproterenol nor (R,R′)-Fen, at 10 μM, elicited an increasein cAMP production in HepG2 cells, whereas cell treatment with theaciculate cyclase activator, forskolin, induced significant accumulationof cAMP (FIG. 1B). Studies have demonstrated that β₂-AR can signal tothe mitogen-activated protein kinases ERK1 and ERK2 independent of afunctional adenylate cyclase coupling. The effect of isoproterenol and(R,R′)-Fen on Akt and ERK1/2 activation was assessed by imunoblottingusing selective antibodies to phosphorylated peptides that correspond tothe active forms of Akt and ERK1/2. Treatment of HepG2 cells with theseβ-agonists induced a time-dependent increase in Akt and ERK activation(FIG. 1C). These results indicate that due to low receptor number,β₂-AR-stimulated adenylyl cyclase activity might be below detectablelevels in this cell line.

The Effects of (R)-Isoproterenol and Fen Analogs on the Proliferation ofHepG2 Cells.

The effect of (R)-isoproterenol, (R,R′)-Fen and selected Fen analogs oncell proliferation was determined in HepG2 cells. Both (R)-isoproterenoland (R,R′)-Fen produced a significant increase in cell proliferation, asassessed by [³H]-thymidine incorporation, with ED₅₀ of 0.40±0.08 μM and1.17±0.37 μM, respectively (Table 1; FIG. 2A). Fen has two chiralcenters and has 4 possible steroisomeric forms, (R,R′), (R,S′), (S,R′)and (S,S′). The effect of the stereochemistry on the proliferativeeffect of Fen was determined using a concentration of 1 μM of eachisomer. The data indicate that all of the isomers induced an increase in[³H]-thymidine incorporation and that stereochemistry had only aquantitative effect on this process with (R,R′)-Fen producing thegreatest increase (51.3%) and (S, S′)-Fen the lowest (9.7%) (Table 1).This result was consistent with the previously reported inhibitoryeffect of Fen stereoisomers on mitogenesis in 1321N1 cells in which theinhibitory potency was (R,R′)>(R,S′)≈(S,R′)>>(S, S′) (see Table 1below). The effect of the change of the N-alkyl methyl group to an ethylmoiety {(R,R′)-ethylFen} and the substitution of an 4′-amino group forthe 4′-hydroxyl group {(R,R′)-aminoFen} were also investigated. Neitheralteration changed the direction of the effect on [³H]-thymidineincorporation and (R,R′)-aminoFen appeared to be 3-fold more active than(R,R′)-Fen with an EC₅₀=0.47±0.09 μM (Table 1; FIG. 2A).

The inventors determined that the incorporation of a naphthyl moietyinto the Fen molecule reduced the potency of the resulting compound, butnot the inhibitory effect on mitogenesis in 1321N1 cells. Further, theopposite effect was observed as (R,R′)-MNF and 1-naphthylFen inhibited[³H]-thymidine incorporation with IC₅₀ values of 0.39±0.09 μM and0.21±0.07 μM, respectively (Table 1; FIG. 2B). The change in thestereochemistry of the chiral center on the N-alkyl portion of the MNFmolecule had no effect on the anti-proliferative response as 1 μMconcentrations of (R,R′)-MNF and (R,S′)-MNF produced equivalentdecreases in [³H]-thymidine incorporation of −59.4% and −68.1%,respectively (Table 1).

TABLE 1 Structures, percent change in thymidine incorporation andIC₅₀/EC₅₀ of fenoterol (Fen) and analogs that were used for this study.

Mitogenesis Inhibition in IC₅₀/EC₅₀ % Change in 1321N1 cells CompoundsR1 R2 (μM) HepG2 cells (IC₅₀ nM)* Fen CH₃

1.17 ± 0.37 (n = 6) (R,R′): 51.3 (R,S′): 19.1 (S,R′): 28.7 (S,S′): 9.7 0.14 ± 0.07  6.09 ± 1.93  6.74 ± 2.18  184.2 ± 26.1 ethylFen CH₃—CH₂

n.d. (R,R′): 50.90 at 10 μM  1.44 ± 0.27 aminoFen CH₃

0.47 ± 0.09 (n = 3) (R,R′): 54.37 1-naphthylFen CH₃

0.21 ± 0.07 (n = 2) (R,R): −67.52  1.57 ± 0.34 4′-methoxy-1- naphthylFenCH₃

0.39 ± 0.09 (n = 6) (R,R′): −59.4 (R,S′): −68.1  3.98 ± 0.28  4.37 ±0.70

(R,R′)-MNF is a full and potent β2-AR agonist in respect to thestimulation of cAMP expression in HEK cells stably transfected withβ₂-AR and in 1321N1 cells, with EC₅₀ of 3.9 nM and 68.9 nM,respectively. Since HepG2 cells displayed substantial sensitivity to(R,R′)-aminoFen (EC₅₀=0.47±0.09 μM) and (R,R′)-MNF (IC₅₀=0.39±0.09 μM)with regard to [³H]-thymidine incorporation, the responsiveness of1321N1 cells to the two compounds was determined and found to bemarkedly lower (FIG. 2C). The specificity of the observed β₂-AR responseto (R,R′)-Fen and (R,R′)-MNF in the HepG2 and 1321N1 cells was testedusing the U87MG cells, which had been previously shown to lack theexpression of active β2-AR. In this cell line, (R,R′)-MNF produced apotent inhibition of cellular proliferation while (R,R′)-Fen had noeffect (FIG. 7).

In the previous study of the effect of (R)-isoproterenol and (R,R′)-Fenon mitogenesis in 1321N1 cells, the studies were conducted usingcomplete medium. In order to determine if the presence of serum or itsabsence significantly influenced the extent of mitogenesis in responseto (R)-isoproterenol and (R,R′)-Fen, the studies were repeated usingboth protocols. The results indicated that HepG2 cells exhibited abetter sensitivity in serum-depleted medium, whereas the sensitivity wasgreater in 1321N1 cells maintained in complete medium (FIG. 2D). Thesedata suggest that there are contrasting mitogenic responses to β₂-ARagonists in HepG2 and 1321N1 cells.

β₂-AR Antagonism does not Inhibit the Anti-Proliferative Action of(R,R′)-MNF while Preventing (R,R′)-Fen's Growth Promoting Effects inHepG2 Cells.

The divergent actions mediated by (R,R′)-Fen and (R,R′)-MNF areconsistent with activation of distinct signaling pathways with oppositeeffects on cell proliferation. To evaluate this, HepG2 cells werepretreated with the β₂ receptor antagonist, ICI 118,551, followed byincubation in the presence of (R,R′)-Fen or (R,R′)-MNF for 24 h. WhileICI 118,551 alone showed no effect on cell proliferation (FIG. 3A), itsaddition significantly blocked (R,R′)-Fen-stimulated mitogenesis (FIGS.3B and 3C). However, the anti-proliferative effect of (R,R′)-MNF wasrefractory to ICI 118,551 pretreatment (FIGS. 3B and 3D).

The ability to hamper the action of (R,R′)-Fen by the co-addition of MNFwas determined. The results showed clearly a mitogenic response in HepG2cells that was intermediate between (R,R′)-Fen and (R,R′)-MNF alone, andthe pretreatment with ICI 118,551 partially restored theanti-proliferative effects of (R,R′)-MNF (FIG. 7, upper panel). However,characteristics of the cell proliferation profile elicited by (R,R′)-Fenin 1321N1 cells and (R,R′)-MNF in U87MG cells were maintained by theco-treatment with (R,R′)-Fen and (R,R′)-MNF (FIG. 7, middle and lowerpanels). Pretreatment with ICI 118,551 blocked (R,R′)-Fen signaling in1321N1 cells while being inactive against the anti-proliferative actionof (R,R′)-MNF in U87MG cells (FIG. 7). These results indicate that theeffects of (R,R′)-Fen and (R,R′)-MNF on cell proliferation are celltype-specific and may require activation of distinct receptors.

(R,R′)-MNF Induces Apoptosis in HepG2 Cells.

The proliferation of HepG2 cells was assessed by flow cytometry analysisusing propidium iodide staining to examine the cell cycle. (R,R′)-Fenproduced no significant alterations of the cell cycle, but (R,R′)-MNFcaused a temporal decrease in the G₂/M- and S-phase cell populations(G2/M: 13.8±1.1% in control versus 10.2±0.9% after 6 h, 14.6±1.8% after12 h and 8.9±1.6% after 24 h; S. 34.7±0.3% in control versus 34.1±0.9%after 6 h. 13.7±1.2% after 12 hand 24.6±4.2% after 24 h) in HepG2 cellstreated with 1 μM (R,R′)-MNF (FIG. 4). The treatment with (R,R′)-MNFalso yielded a time-dependent increase in the number of sub-G₁ events,reaching a maximum of 21.5±0.7% by 12 hours (FIG. 4, bottom, rightpanel). No significant increase in sub-G₁ events was observed when cellswere treated with (R,R′)-Fen (1 μM) for up to 24 hours.

Sub-G₁ events occur when cells have proceeded to the late stage ofapoptosis or are already dead. To directly measure apoptosis, flowcytometry analysis with Annexin V/PI staining was carried out in HepG2cells. The percentage of apoptotic cells induced by a 24-hour treatmentwith (R,R′)-MNF (1 μM) increased 5.7-fold compared to the control(P<0.01). However, (R,R′)-Fen treatment markedly reduced apoptosis whencompared to control untreated cells (FIG. 5).

Role of Cannabinoid Receptors in the Control of Cell Proliferation of(R,R′)-MNF and (R,R′)-Fen.

Whether the regulation of mitogenesis in response to (R,R′)-Fen and(R,R′)-MNF occurs through cannabinoid receptor signaling mechanisms wasexamined. The mRNA levels of CB1R and CB2R were determined by RT-PCR inHepG2, 1321N1 and U87MG cells (FIG. 6A, primers provided below in Table2). The results indicated that HepG2 and U87MG cells expressed CB1R andCB2R, whereas 1321N1 cells had no detectable levels of CBR mRNAs. Potentregulatory effects of synthetic cannabinoid compounds were observed incells treated with (R,R′)-Fen and (R,R′)-MNF as compared with controls.Similar to (R,R′)-MNF, treatment of HepG2 cells with the cannabinoidreceptor agonist, WIN55,212-2 (1 μM), reduced cell proliferation andcanceled out the growth-promoting action of fenoterol (FIG. 6B). AM251and AM630 are synthetic inverse agonists for CB1R and CB2R,respectively. Cell pretreatment with AM251 or AM630 had no impact on themitogenic responses of (R,R′)-Fen (FIG. 6C), indicating that basal-levelactivity of these two cannabinoid receptors does not play a major rolein the proliferative action of (R,R′)-Fen. However, preincubation withAM251 or AM630 completely inhibited the anti-proliferative effects of(R,R′)-MNF in HepG2 cells (FIG. 6C), which is consistent with theinvolvement of cannabinoid receptors in (R,R′)-MNF signaling. In supportof this hypothesis, 1321N1 cells, which are (R,R′)-MNF unresponsive,were refractory to cannabinoid receptor ligands when added alone orcombined with (R,R′)-Fen (FIGS. 8A and 8B). However, theanti-proliferative effects of MNF were partially blocked by AM251 andAM630 in the β2-AR-deficient, MNF responsive U87MG cells (FIGS. 8C and8D).

TABLE 2List of PCR primers and assay conditions. Each of the references listed in the below Table is hereby incorporated by reference in its entirety.Initial Amplification (35 cycles) Human Denatura- Denatura- Final GenePrimers tion tion Annealing Extension extension CB1 F: 5′- 95° C./ 94°C./ 57° C./ 72° C./ 72° C./ (SEQ ID CGTGGGCAGCCT 5 min 30 sec 30 sec1 min 5 min NOs: 1 GTTCCTCA and 2, R: 5′- resp.) CATGCGGGCTTG GTCTGG CB2F: 5′- 95° C./ 94° C./ 57° C./ 72° C./ 72° C./  (SEQ ID CGCCGGAAGCCC5 min 30 sec 30 sec 1 min 5 min NOs: 3 TCATACC and 4) R: 5′-CCTCATTCGGGC CATTCCTG GAPDH F: 5′- 94° C./ 94° C./ 53° C./ 72° C./ 72°C./ (SEQ ID ACCACAGTCCATGCCATC 4 min 1 min 1 min 1 min 10 min NOs: 5R: 5′- and 6, TCCACCACCCTGTTGCTG resp.) β2AR F: 5′- 94° C./ 94° C./ 58°C./ 72° C./ 72° C./ (SEQ ID CATGTCTCTCATCGTCCTG 6 min 1 min 30 sec 1 min5 min NOs: 7 GCCA and 8, R: 5′- resp.) CACGATGGAAGAGGCAAT GGCA

The present Example demonstrates that treatment of HepG2 cells with(R,R′)-Fen led to increased cellular proliferation. ICI 118,551 blockedthis effect indicating the involvement of β₂-ARs. However, neither(R,R′)-Fen- or isoproterenol induced formation of cAMP in HepG2 cells,although treatment with forskolin demonstrated that the cells expressedfunctional adenylate cyclase (FIG. 1). These results support twopossibilities to explain the lack of effect of β₂-AR agonists on cAMPaccumulation: either the β₂-ARs are at a low enough level that they donot significantly increase cAMP, or the β₂-ARs in the HepG2 cell lineare poorly coupled to the stimulatory Gα protein and suggest potentialinteractions with other signaling intermediates that promote cellgrowth. The present results demonstrate that treatment of HepG2 cellswith (R,R′)-Fen or isoproterenol activated the PI3-kinase/Akt and ERKpathways.

Previous studies on the effect of the stereochemistry of Fen onβ₂-AR-associated stimulation of cAMP accumulation and inhibition ofmitogenesis in 1321N1 cells have demonstrated that changes in thespatial configurations at the molecule's two chiral centers producesonly quantitative changes in these properties. A similar effect ofstereochemistry was observed in the HepG2 cells as all of the Fenstereoisomers produced an increase in [³H]-thymidine incorporation(reported as % change) with (R,R′)>>(S,R′)≈(R,S′)>>(S,S′) (Table 1). Theeffect of structural changes in the (R,R′)-Fen molecule was investigatedby increasing the steric bulk at the chiral center on the N-alkylportion of the molecule, (R,R′)-ethylFen, and by changing the hydrogenbonding properties of the 4′-substituent, (R,R′)-aminoFen. Both analogsincreased [³H]-thymidine incorporation in HepG2 cells to the same extentas (R,R′)-Fen (Table 1 and FIG. 2A), suggesting that when the Fenmolecule contains a 4′-substituted phenyl ring, the compound stimulates[³H]-thymidine incorporation in HepG2 cells and that the stereochemistryof the molecule influences this effect, but does not qualitativelychange it. A full structure-activity relationship study has beeninitiated and the results will be reported elsewhere.

The inventors demonstrated that naphthylfenoterol (NF) analogs of Fenproduced by the substitution of a naphthyl moiety for the phenyl ring onthe N-alkyl portion are full and potent 13₂-AR agonists with respect tothe stimulation of cAMP expression in HEK cells stably transfected withβ2-AR (HEK-β2-AR); for example, EC_(50cAMP) of (R,R′)-1-NF and(R,R′)-MNF were 12.5 and 3.9 nM, respectively. As was observed with(R,R′)-Fen, (R,R′)-1-NF and (R,R′)-MNF also inhibited mitogenesis in1321N1 cells although the IC₅₀ values were ≥10-fold higher than(R,R′)-Fen (Toll et al., J Pharmacol Exp Ther 336:524-32, 2011). Asimilar quantitative effect was expected when HepG2 cells were incubatedwith (R,R′)-1-NF and (R,R′)-MNF, that is, a weaker stimulation of[³H]-thymidine incorporation. However, a qualitative different effectwas observed as (R,R′)-MNF and (R,R′)-1-NF inhibited [³H]-thymidineincorporation with IC₅₀ values of 0.39±0.09 μM and 0.21±0.07 μM,respectively (Table 1 and FIG. 2B). In addition, unlike Fen, a change inthe stereochemistry of the chiral center on the N-alkyl portion of theMNF molecule had no effect on the anti-proliferative response (Table 1).

Since the Fen and NF analogs used in this study are β2-AR agonists, apotential explanation for the effect produced by the substitution of anaphthyl ring for a phenyl ring is “ligand-directed signaling” or“biased agonism.” It has been demonstrated that the β2-AR binds ligandsin multiple conformations and that binding to different receptorconformations can lead to differences in signal transduction. In respectto the Fen and NF molecules, initial Comparative Molecular FieldAnalysis (CoMFA) studies of the interaction of the Fen analogs with theβ₂-AR have indicated that the naphthyl substituent of the NFs moleculescan interact with the β2-AR through a series of π-π and π-hydrogen bondinteractions unavailable to the phenyl moiety on the Fen molecule.However, the direct association of the binding of the NF analogs and thedecrease in [³H]-thymidine incorporation appears doubtful as theselective pharmacological β₂-AR antagonist, ICI 118,551, failed to blockthe anti-proliferative action of (R,R′)-MNF (FIG. 3) and treatment ofthe β2-AR-negative U87MG cells with (R,R′)-MNF causes a marked reductionin cell growth while (R,R′)-Fen had no effect (FIG. 7). This data doesnot eliminate the possibility that NFs bind to and stabilize aconformation of the β₂-ARs expressed in HepG2 cells that is distinctfrom the conformation stabilized by (R,R′)-Fen, but it suggests that ifthis occurs it does not result in the initiation of a downstreamsignaling cascade that effects cellular growth.

Pharmacological evidence disclosed herein indicates that (R,R′)-MNFmediates its anti-proliferative effects through activation of the CBreceptors. On one hand, this action of (R,R′)-MNF was reproduced by WIN55,212-2 in HepG2 and U87MG cells, and the combination (R,R′)-MNF plusWIN 55,212-2 showed a lack of additive effect. On the other hand,selective inhibition of the CB1 and CB2 receptors showed suppression of(R,R′)-MNF signaling. The finding of a lack of effect of the WIN55,212-2-mediated actions on cell growth in 1321N1 cells may beexplained by there being substantially more CBR expression in HepG2 andU87MG cell lines than in 1321N1 cells. Moreover, the present resultsdemonstrate that the (R,R′)-Fen-mediated increase in HepG2 cellproliferation was neutralized by WIN 55,212-2, possibly indicating thatstimulation of G_(i)-linked CBRs in response to WIN 55,212-2 inhibitscell growth primarily by negatively targeting the PI3-kinase/Akt and/orERK pathways. Taken together, these findings indicate a complex celltype-specific involvement of CB receptors in the anti-mitogenic andproapoptotic activities of (R,R′)-MNF through a mechanism that does notrequire β₂-AR activation.

Example 3 Characterization of Cannabinoid Receptor Modulation

This example describes a series of studies performed to furthercharacterize the ability of fenoterol and disclosed fenoterol analogs tomodulate cannabinoid receptor activity.

To identify the type of CB receptor mediating the (R,R′)-MNF-inducedresponse, the effect of MNF on TocriFluor 1117 (T1117, TocrisBioscience) uptake in HepG2 cells was evaluated. T1117 is a fluorescentform of the cannabinoid CB1 receptor inverse agonist AM251, which bindsGPR55 with high affinity, but has modest binding with CB1 receptors andno interaction with CB2 receptors. HepG2 cells were incubated withfenoterol (1 μM), MNF (1 μM) or AM251 (10 μM) for 1 hour followed byaddition of T1117 (0.1 μM). As illustrated in FIG. 9, T1117 uptake wasdramatically reduced in cells treated with MNF or AM251 prior to T1117as compared to either vehicle or fenoterol. These studies indicate thatthe MNF is capable of modulating GPR55.

Example 4 In Vitro Metabolic Stability of MNF

This example describes the metabolic stability of MNF on human and ratliver microsomes.

FIG. 10 presents the data from the in vitro metabolic stability study inwhich MNF was incubated (in duplicate) with active and heat-inactivatedhuman (HLM) and rat (RLM) liver microsomes and cofactors at 37° C.Aliquots were removed at 0, 15, 30 and 60 minutes and the amount of testcompound (MNF) remaining at each time point was determined by LC-MS/MS.Although the results indicated that MNF is somewhat unstable under theincubation conditions, based on the decrease in concentration in theheat-inactivated microsome controls, the results show a greater decreasein MNF levels when incubated with rat and human liver microsomes.

FIG. 11 demonstrates the results from the co-incubation of MNF, 1 or 10μM, with a cocktail of model CYP substrates, human liver microsomes andCYP cofactors for 20 minutes at 37° C. Incubations containing known CYPinhibitors were also included as positive controls. Formation of themodel substrate metabolites was measured by LC-MS/MS and compared tocontrol incubations (incubation of substrates with microsomes andcofactors, no test articles or inhibitors). CYP activity in presence oftest compound (MNF) that was less than 70% of control was consideredsignificant. MNF (10 μM) inhibited CYP2D6 and CYP3A4 to less than 70% ofthe control. Further, the primary metabolite was determined to beO-demethylated-MNF. These studies indicate that MNF alters themetabolism of other drugs or endogenous compounds that are substratesfor CYP2D6 and/or CYP3A4 isoforms, when present in the plasma or liverat 10 μM or higher.

Example 5 In Vivo Distribution and Clearance after IV Administration

To determine plasma and brain tissue concentrations of MNF, theconcentrations of MNF and its metabolites in plasma and brain tissuesamples obtained from male Sprague-Dawley rats were determined afteradministration of a single IV dose of 10 mg/kg of MNF. The assays wereconducted using an Eclipse XDB-C₁₈ guard column (4.6 mm×12.5 mm) and anAtlantis HILIC column (150×2.1 mm ID, 5 mm). The mobile phase consistedof water containing 0.1% formic acid as Component A and acetonitrile ascomponent B. A linear gradient was run as follows: 0 minutes 95% B; 5minutes 60% B; 6 minutes 80% B; 10 minutes 95% B at a flow rate of 1.0ml/minutes. The total run time was 15 minutes per sample. Identificationand quantification of the analytes was accomplished using an API-4000LC-MS/MS in positive electrospray ionization mode and data was acquiredemploying multiple reaction monitoring (MRM) and the following MRMtransitions: MNF (369-200); MNF-Gluc (545-200).

FIG. 12 illustrates the analysis of a plasma sample obtained 30 minutespost-W administration of 10 mg/kg MNF. In the insert of FIG. 12, MNF andGluc-MNF are shown with no interfering peaks being present in thecontrol plasma matrix. FIG. 13 illustrates the analysis of brain tissueobtained 30 minutes post-W administration of 10 mg/kg MNF. The peak at6.39 minutes is an unidentified compound present in control brain matrix(see insert of FIG. 13). FIG. 14 is a MNF time-course in plasma andbrain of Sprague-Dawley male rats and the calculated pharmacokineticparameters are presented in Table 3 below.

TABLE 3 Calculated pharmacokinetic parameters for MNF time-course inplasma and brain. AUC_(last) AUC_(inf) Cl Rat t_(1/2) (hr) C_(max)(ng/ml) C⁰ (ng/ml) (hr · ng/ml) (hr · ng/ml) V (l/kg) (ml/hr/kg) 1 9.81780.7 3033.4 3582.2 4103.6 34.3 2436.9 2 18.6 2304.9 4131.4 4070.75335.2 50.4 1874.4 3 13.0 1383.4 2340.7 2905.1 3695.2 50.6 2706.2 Mean13.8 1823.0 3168.5 3519.3 4378.0 45.1 2339.1 SD 4.5 462.2 903.0 585.3853.7 9.4 424.5 Abbreviations Elimination phase half life = t_(1/2);maximum plasma concentration observed = C_(max); plasma concentrationextrapolated to time 0 = C⁰; area under the plasma versus time curve tolast time point and extrapolated to infinity = AUC_(last), AUC_(inf);apparent volume of distribution = V; whole body clearance = Cl.

Ten mg/kg MNF was administered IV to male Sprague-Dawley rats and theplasma concentrations of MNF were determined between 10 minutes and 24hours post administration with the levels measured in 3 animals per timepoint. In the same study, the concentration of MNF in brain tissue wasmeasured between 10 and 60 minutes after administration with 3 animalsper time point. The MNF concentration in brain tissue was 200 ng/mgtissue at 10 minutes after administration and peaked at 30 minutes at800 ng/mg tissue. The relative distribution between the concentration ofMNF in blood (measured as ng/ml) and brain tissue (measured as ng/mgtissue) was 0.2 at 10 minutes and 1.0 at 30 minutes and 60 minutesreflecting an equivalent distribution between both the central andperipheral body compartments. This analysis indicates that significantlevels of the drug can be quantified in the brain tissue 10 minutesafter administration and that the concentration peaks at 30 minutesafter dosing, at which time it parallels the plasma concentration andthat at 60 minutes, the brain tissue concentration continues to parallelthe plasma concentration. Further, the apparent volume of distribution(V) is high indicating extensive tissue distribution.

These studies demonstrate that MNF is capable of passing through theblood brain barrier and that administration of such compound as well aslikely other related fenoterol analogues and/or fenoterol by IV is aneffective means of delivering these compounds to the brain, such as totreat a brain tumor regulated by CB receptor activity.

Example 6 MNF has No Significant Negative Effects on Central NervousSystem Function

This example demonstrates that MNF has no significant negative effectson central nervous system function.

A single dose escalation study was performed to select dose levels for7-day repeat dose study with IV administration in Sprague-Dawley rats.Dose levels were 0, 5, 10 and 25 mg/kg. Toxic effects were minimal inthis study (see Tables 4 and 5 below).

TABLE 4 7-Day Repeat Dose Range Finding Study of MNF in Sprague-Dawleyrats. Endpoints: body weight change; clinical observations; clinicalpathology; gross necropsy; and histopathology. Number of Dose Dose DoseNumber of Animals Test Level Volume Concentration^(b) Animals MainRecovery Article Vehicle (mg/kg) (ml/kg) (mg/ml) Groups^(a) Groups^(a)MNF 0.9% Sodium Chloride 0 5 0 3M/3F 3M/3F for injection, USP SRI-128380.9% Sodium Chloride 2.5 5 0.5 3M/3F 3M/3F for injection, USP SRI-128380.9% Sodium Chloride 10 5 2 3M/3F 3M/3F for injection, USP SRI-128380.9% Sodium Chloride 25 5 5 3M/3F 3M/3F for injection, USP

TABLE 5 Evaluation of MNF study shown in Table 4. Species/ ClinicalClinical Gross Nectopsy Histo- Strain Study Design ^(a) Body Weight ^(b)Observation ^(c) Pathology ^(d) Observation ^(e) pathology Rat/Escalation: Body weight for Slight Increases~2-fold Discolored red:Evaluation Sprague 0, 2.5, 5, 10 or treated rats was hypoactivity: WBC,% Neut, thymus, in progress Dawley 25 mg/kg comparable all treated rats% RET/REA, and mandibular lymph 7-Day: with control post dose PLC node0, 5, 10 or 25 animals Necrosis: Discolored black mg/kg/day high-dose ornecrosis: tail ^(a) Dosing regimen (5 ml/kg in 0.9% saline): singleintravenous (iv) injection via the lateral tail vein (escalation study);single daily iv dose with a 7 day recovery period (7-day study) ^(b)Body weight collected: Day 1 for escalation dose calculation; Day 1, Day3, and Day 8 (Main and Recovery) and Day 14 (Recovery) for 7-day repeatstudy. ^(c) Observations seen during 7-Day repeat study: performeddaily; hypoactivity observed~5 minutes post-dose and subsided~1 hourpost-dose; necrosis observed at injection site for males and females.^(d) Hematology and clinical chemistry evaluations performed Day 8 andDay 14; increases seen in high-dose males and females at interim andterminal sacrifice appear to be associated with necrosis at dose site.^(e) Observations seen at interim (Day 8) and terminal (Day 14) timepoints.

Additional studies were performed which further evaluated the effect ofMNF on central nervous system functions. In these studies, MNF was foundto not have any significant negative effects on the central nervoussystem (FIG. 15) as compared to tetrahydrocannabinol (FIG. 16).

Example 7 (R,R′)-MNF and (R,R′)-4-Methoxyfenoterol (MF) are PotentInhibitors of Liver, Colon and Lung Cancer Cell Growth

This example demonstrates that (R,R′)-MNF and (R,R′)-4-methoxyfenoterol(MF) are potent inhibitors of liver, colon, and lung cancer cell growth.

The effect of (R,R′)-MF and (R,R′)-MNF on the growth of a variety oftumor cells was evaluated. An IC₅₀ value of less than 30 μM wasconsidered to be an effective inhibitor of tumor cell growth whereas anIC₅₀ value of less than 50 μM indicates potential activity. Asillustrated in Tables 6 and 7 below, MNF was a potent inhibitor of livercancer cell growth, lung cancer cell growth, colon cancer cell growth,prostate cancer cell growth, CNS cancer cell growth, and non-small celllung cancer (NSCLC).

These studies indicate that MNF and MF are capable of inhibitingadditional types of cancer growth, including liver, lung and coloncancer. One of skill in the art will appreciate that they also providesupport for using other fenoterol analogues (such as othernaphthylfenoterol analogues), to reduce tumor growth, including treatingcancer, such as liver, lung and colon cancer, in additional subjects,including humans.

TABLE 6 Effect of MNF and MF on various tumor cells. IC50 (uM) seedingIn vivo RR- RR- T3 # Type Name n/well imaging MF MNF Doxorubicin % CVT3/T0 None 1 Pancreatic CAPAN 750 N.E Not 0.23 5.16 1.71 CNS C Regress.added 2 Pancreatic PANC-1 1125 N.E. 33.38 2.97 12.69 1.73 C 3 Liver CHEPG2 750 Caliperls N.E. 28.82 0.28 7.68 3.11 4 Liver C HEP3B 750 >50 uM14.71 0.43 4.55 2.42 NCI60 5 Prostate C Du-145 750 >50 uM 34.76 0.383.34 4.39 Subpanel 6 Prostate C PC3 1125 >50 uM 28.06 0.91 2.18 4.08 7Breast C MCF7 2250 N.E. 37.19 0.30 3.23 2.51 8 Breast C MDAM 1500 SRIstock N.E. 33.23 0.68 3.28 3.94 B231 9 NSCLC H460 750 >50 uM 11.59 0.022.90 12.78 10 NSCLC A549 750 >50 uM 23.14 0.11 3.27 7.96 11 Colon C HT29375 >50 uM 10.71 0.26 5.85 12.46 12 Colon C HCT116 375 >50 uM 12.91 0.065.75 10.87 13 CNS U251 375 N.E. 26.67 0.17 2.48 4.47 CNS 14 CNS U87 1125Caliperls N.E. 25.72 0.21 10.16 3.71 Panel 15 CNS GL261 375 Caliperls N.E.? 21.32 0.17 7.54 4.61 16 CNS LN-18 1125  N.E.? 29.74 0.53 2.766.25 17 CNS A172 750 ?  N.E.? 30.48 0.46 5.08 2.77 18 CNS LN-229 750? >50 uM 26.77 0.61 2.23 3.90 19 CNS U118 1125 ? >50 uM 37.04 0.81 2.662.43 20 CNS 1321N 1125 ? TBD TBD TBD TBD TBD

TABLE 7 Effect of MNF and MF on various tumor cells. seeding IC50 (uM)Name n/well RR-MF RR-MNF Doxorubicin Vc CV Tr/Tz^(a) Note HEP3B 750 >50uM 14.71 0.43 4.55 2.42 384 well CTG 3 days treatment-1 (Liver >50 uM12.14 0.35 8.65 2.22 384 well CTG 3 days treatment-2 Cancer) 4275 >50 uM13.53 0.52 6.56 1.74  96 well CTG 2 days treatment 2.35 10.03 0.02 13.38NA^(b)  96 well Thy 2 days treatment >50 uM 28.30 NF^(c) 4.40 1.19  96well CTG 1 day treatment 2.40 10.69 0.04 14.66 NA^(b)  96 well Tye 1 daytreatment PC3 1125 >50 uM 28.06 0.91 2.18 4.08 384 well CTG 3 daystreatment-1 (Prostate 6412.5 >50 uM 20.36 0.93 3.10 5.89  96 well CTG 2days treatment Cancer) >50 uM 10.84 0.28 13.74 NAb  96 well Thy 2 daystreatment >50 uM 34.51 3.02 4.52 2.66  96 well CTG 1 day treatment >50uM 10.95 0.04 9.16 NA^(b)  96 well Tye 1 day treatment NCIH460 750 >50uM 11.59 0.02 2.90 12.78 384 well CTG 3 days treatment-1 (NSCLC) >50 uM17.68 0.11 3.10 9.71 384 well CTG 3 days treatment-2 4275 >50 uM 15.400.12 7.82 5.26  96 well CTG 2 days treatment >50 uM 21.03 0.03 17.96NA^(b)  96 well Thy 2 days treatment >50 uM 38.92 0.31 3.10 1.30  96well CTG 1 day treatment >50 uM 25.08 0.09 16.10 NA^(b)  96 well Tye 1day treatment HCT116 375 >50 uM 12.91 0.06 5.75 10.87 384 well CTG 3days treatment-1 (Colon >50 uM 12.20 0.21 5.39 13.13 384 well CTG 3 daystreatment-2 Cancer) 2137.5 >50 uM 10.54 0.23 6.01 8.30  96 well CTG 2days treatment >50 uM 10.97 0.14 6.79 NA^(b)  96 well Thy 2 daystreatment >50 uM 18.96 0.21 5.34 2.15  96 well CTG 1 day treatment >50uM 23.75 0.12 9.90 NA^(b)  96 well Tye 1 day treatment HT29 375 >50 uM10.71 0.26 5.85 12.46 384 well CTG 3 days treatment-1 (Colon 2137.5 >50uM 11.65 0.52 8.63 6.11  96 well CTG 2 days treatment Cancer) >50 uM19.57 0.12 44.53 NA^(b)  96 well Thy 2 days treatment >50 uM 30.40NF^(c) 3.84 1.56  96 well CTG 1 day treatment >50 uM 12.98 0.11 17.05NA^(b)  96 well Tye 1 day treatment LN229 750 >50 uM 26.77 0.61 2.233.90 384 well CTG 3 days treatment-1 (CNS 4275 >50 uM 15.69 1.70 1.802.20  96 well CTG 2 days treatment Cancer) >50 uM 10.37 0.07 12.20NA^(b)  96 well Thy 2 days treatment >50 uM 29.32 NF^(c) 2.70 1.37  96well CTG 1 day treatment >50 uM 9.64 0.10 12.09 NA^(b)  96 well Tye 1day treatment VcCV Percent Variance of control wells on day of readingcell growth measured by comparing control wells before and aftercompound Tr/Tz^(a) treatment; NA^(b) not applicable NF^(c) no optimumfitting

Example 8 Antitumor Activity of (R,R′)-4-Methoxy-1-Naphthylfenoterol ina Rat C6 Glioma Xenograft Model in the Mouse

This example demonstrates antitumor activity of MNF in a rat C6 gliomaxenograft model in the mouse.

(R,R′)-4-methoxy-1-naphthylfenoterol (MNF) inhibits in vitroproliferation of several types of cancer cell lines. In this example,the in vivo antitumor effects of MNF were evaluated using rat C6 gliomacells implanted subcutaneously into the lower flank of 5 week-oldNMRI/Nude female Swiss mice. Three days after the inoculation, the micewere subjected to intraperitoneal injections of saline or MNF (2 mg/kg)for five days per week for two weeks. Tumor volumes were measuredeveryday using slide calipers. At the end of the study, animals weresacrificed and tumors were collected for cDNA microarray, quantitativeRT-PCR and immunoblot analyses. Significant reduction in mean tumorvolumes was observed in mice receiving MNF when compared with thesaline-treated group (p<0.001, n=17-19). Clusters in expression of genesinvolved in cellular proliferation were identified, as well as molecularmarkers for glioblastoma that were significantly downregulated in tumorsof MNF-treated mice as compared to saline-injected controls. Theefficacy of MNF against C6 glioma cell proliferation in vivo and invitro was accompanied by marked reduction in the expression of cellcycle regulator proteins. This study is the first demonstration ofMNF-dependent chemoprevention in a glioblastoma xenograft model andprovides a mechanism for its anticancer action in vivo.

Materials and Methods

Materials.

(R,R′)-4-methoxy-1-naphthylfenoterol (MNF) was synthesized as describedherein and previously (Jozwiak et al., J Med Chem 50: 2903-2915, 2007;Jozwiak et al., Bioorg Med Chem 18:728-736, 2010; each of which isincorporated by reference in its entirety). Dulbecco's modified EagleMedium (DMEM), trypsin solution, phosphate-buffered saline (PBS), fetalbovine serum (FBS), 100× solution of L-glutamine (200 mM), andpenicillin/streptomycin (a mixture of 10,000 units/ml penicillin and10,000 μg/ml streptomycin) were obtained from Quality Biological(Gaithersburg, Md., USA).

Cell Culture.

The rat-derived C6 glioma cell line was obtained from the American TypeCulture Collection (Manassas, Va.). The cells were routinely maintainedin DMEM supplemented with L-glutamine, 1% penicillin/streptomycinsolution, and 10% FBS in a humidified CO2 incubator at 37° C.

3H-Thymidine Incorporation.

C6 glioma cells were seeded in 12-well plates at ˜5×104 cells/well andincubated for 24 hours followed by a second 24-hour incubation withvarious concentrations of MNF. Radiolabeled thymidine (10 Ci/mmol;PerkinElmer Life and Analytical Sciences, Waltham, Mass.) was added at 1μCi per well for 16 hours and its incorporation into DNA was thenmeasured. Each treatment group was performed in triplicate and threeindependent studies were carried out.

C6 Tumor Xenograft in Mice.

In order to assess the ability of MNF to induce regression of tumorgrowth in vivo, rat C6 glioma cells were trypsin-collected at confluencyand were used to generate tumor xenografts. Athymic female nude mice(SWISS nu+/nu+) were obtained from Charles Rivers (L'Arbresle, France)and maintained under pathogen-free conditions with a 12 hours light/12hours dark cycle. Animals were fed ad libitum with normal chow(supplier). Athymic nude mice were inoculated subcutaneously with 100 μlof culture medium containing 0.5×106 C6 glioma cells in the left flankand then were randomly divided into two groups of 10 animals each.Starting 3 days after cell inoculation, mice received dailyintraperitoneal injection (10 μl.g-l body weight) of vehicle or MNF (2mg·kg-l) in 100 μM ascorbic acid in saline (vehicle) five days a weekfor 19 days. Animal survival was monitored daily, and tumor size wasdetermined with the use of a caliper to measure the length (a) and width(b) and estimated as 4/3π×r12×r2, where r1 is the smaller and r 2 thelarger radius. The mice were monitored up to 19 days after MNF injectionor euthanized earlier if the tumor size was superior to 2 cm3 or themouse was lethargic, sick and unable to feed, which caused the bodyweight to drop below 20% of initial weight. The mice were euthanized bycervical extension, and tumor masses were removed, weighed and washedwith cold PBS before being snap-frozen in liquid nitrogen. A second setof studies with 8-9 animals in both groups was repeated.

Evaluation of MNF Accumulation In Vivo in C6 Glioma Tumors.

The accumulation of MNF in vivo in C6 tumor xenografts in athymic micewas assessed in comparison with vehicle-treated tumor-bearing animals.The frozen tumor samples were thawed, and then homogenized. Theconcentration of MNF and its metabolites was determined by HPLC followedby LC-MS/MS. In brief, the assays were conducted using an EclipseXDB-C18 guard column (4.6 mm×12.5 mm) and an Atlantis HILIC column(150×2.1 mm ID, 5 mm). The mobile phase consisted of water containing0.1% formic acid as component A and acetonitrile as component B. Alinear gradient was run as follows: 0 minutes 95% B; 5 minutes 60% B; 6minutes 80% B; 10 minutes 95% B at a flow rate of 1.0 ml/minute. Thetotal run time was 15 minutes per sample. Identification andquantification of the analytes was accomplished using an API-4000LC-MS/MS in positive electrospray ionization mode and data was acquiredemploying multiple reaction monitoring (MRM) and the following MRMtransitions: MNF (369-200); MNF-Gluc (545-200). Tumor tissues fromvehicle-injected mice were used as controls.

Analysis of Gene Expression in Rat C6 Glioma Xenografts.

Total RNA was isolated from rat C6 glioma xenografts harvested fromvehicle and MNF-treated mice (n=3 per group, cohort 1). This analysiswas repeated in a second cohort of animals (n=3 per group, cohort 2).Total cellular RNA was extracted using an RNeasy plus mini kit (QIAGEN,Valencia, Calif.), and its quality was assessed using an AgilentBioAnalyzer using RNA 6000 Nano Chips (Agilent Technologies, SantaClara, Calif.). Transcriptional profiling was determined using IlluminaSentrix BeadChips (Illumina, San Diego, Calif.). Total RNA was used togenerate biotin-labeled cRNA with the Illumina TotalPrep RNAAmplification Kit. In short, 0.5 ug of total RNA was first convertedinto single-stranded cDNA with reverse transcriptase using an oligo-dTprimer containing the T7 RNA polymerase promoter site and then copied toproduce double-stranded cDNA molecules. The double-stranded cDNA wascleaned and concentrated with the supplied columns and used in anovernight in-vitro transcription reaction where single-stranded RNA(cRNA) was generated incorporating biotin-16-UTP. A total of 0.75 μg ofbiotin-labeled cRNA was hybridized at 58° C. for 16 hours to Illumina'sSentrix Rat Ref-12 Expression BeadChips. Each BeadChip has ˜22,000well-annotated RefSeq transcripts with approximately 30-fold redundancy.The arrays were washed, blocked and the labeled cRNA was detected bystaining with streptavidin-Cy3. Hybridized arrays were scanned using anIllumina BeadStation 500× Genetic Analysis Systems scanner and the imagedata extracted using Illumina's GenomeStudio software, version 1.6.1.For statistical analysis, the expression data were filtered to includeonly probes with a consistent signal on each chip and an Illuminadetecton p value <0.02.

Correlation analysis, sample clustering analysis and principal componentanalysis was performed to identify/exclude any possible outliers. Theresulting dataset was next analyzed with DIANE 6.0, a spreadsheet-basedmicroarray analysis program using value statistics for Z-Scorereliability below 0.05; and mean background-corrected signal intensitygreater than zero.

Gene set enrichment analysis use gene expression values or geneexpression change values for all of the genes in the microarray.Parametric analysis of gene set enrichment (PAGE) was used using theWEB-PAGE GSA tool for gene set analysis. Gene Sets include the MSIGdatabase, Gene Ontology Database, GAD human disease and mouse phenotypegene sets were used to explore functional level changes. Gene-geneinteraction was also analyzed using the INGENUITY® Pathway Analysis(IPA) system (INGENUITY® Systems).

Total RNA Extraction, cDNA Synthesis and qRT-PCR Analysis.

Total RNA (including the DNase treatment step) was isolated from frozentumor tissues using the RNeasy mini kit (Qiagen, Valencia, Calif.). RNAconcentration and quality was measured using the NanoDropspectrophotometer (NanoDrop Technologies, Wilmington, Del.).Subsequently, 2 μg total RNA was reverse-transcribed to cDNA using theqSCRIPT™ cDNA SuperMix (Quanta Biosciences, Gaithersburg, Md.).Quantitative real-time PCR (qRT-PCR) reactions were performed tovalidate the expression of 6 genes that were selected from themicroarray analysis. The reactions were carried out with SYBR® Green PCRmaster mix on an ABI Prism 7300 sequence detection system (AppliedBiosystems) using commercially available target probes for Sox4, Olig1,Galnt3, Cdkn3, Ccna2, and Bub1b (PrimeTime qPCR Assays and Primers, IDTDNA Technologies, Coralville, Iowa). The data was analyzed using the2-ΔΔCt method with Gapdh and vehicle-treated tumors as internalcontrols. Controls consisting of reaction mixture without cDNA werenegative in all runs.

Western Blot Analysis.

Frozen tumor tissues were lysed with radioimmune precipitation buffercontaining EGTA and EDTA (Boston BioProducts, Ashland, Mass.)supplemented with a phosphatase inhibitor cocktail (EMB-Calbiochem), andprotease inhibitor cocktail (Sigma-Aldrich), according to standardprotocols. Equal amounts of protein from the clarified lysates wereseparated by SDS-polyacrylamide gel electrophoresis under reducingconditions (Invitrogen, Carlsbad, Calif.), and electrotransferred ontopolyvinylidene difluoride membranes using the iBlot system (Invitrogen).Western blots were performed according to standard methods, whichinvolved blocking the membrane in 5% non-fat milk, followed bysequential incubation method with the primary antibody of interest andsecondary antibody conjugated with the enzyme horseradish peroxidase.The detection of immunoreactive bands was performed by chemiluminescenceusing the ECL Plus Western Blotting Detection System (GE Healthcare,Piscataway, N.J.). Quantitation of the protein bands was done by volumedensitometry using ImageJ software (National Institutes of Health,Bethesda, Md.). Primary antibodies used in this study were raisedagainst cyclin A (sc-751, 1:500 dilution; Santa Cruz Biotechnology,Inc., Santa Cruz, Calif., USA), cyclin D1 (sc-8396; 1:500 dilution;Santa Cruz), and β-actin (mouse; 1:10000 dilution; Abcam, Cambridge,Mass., USA). Detection of Hsp90 with a monoclonal antibody (1:1000;Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) was carried out tocontrol for equal protein loading.

Statistical Analysis.

Western blot data form both sets of tumor tissues were analyzedtogether. The Shapiro-Wilk test was used to assess if the values(protein expression levels) followed a Gaussian distribution. Outlierswere removed from further analysis as they were preventing thepopulation to pass the normality test. The results from rat C6 cells inculture were analyzed using the Student t-test. Repeated two-wayanalysis of variance (ANOVA) was used to compare induction of changes asa function of time. Data were expressed as means±standard error of themean (SEM) and were considered significant when the p value was lessthan 0.05.

Results

MNF Reduces Tumor Cell Proliferation.

When cell proliferation assay was performed using the rat C6 glioma cellline, a potent growth inhibition was observed in response to MNF with aIC₅₀ of ˜1.0 nM (FIG. 18A). The effect of the β-AR agonistsisoproterenol and Fen on cell proliferation was compared to that of MNFin the absence and presence of the selective β2-AR blocker, ICI-118,551(FIG. 18B). Both isoproterenol and Fen elicited weak 10-15% inhibitionof C6 cell growth when used at 20 nM, and the same concentration of MNFcaused a significant 54.3±1.2% reduction in mitogenesis (n=4, P<0.001).The addition of ICI-118,551 did not block the antiproliferative effectof MNF while impeding isoproterenol and Fen signaling (FIG. 18B). C6glioma cells express both β2-AR and CB receptors, and the cellularactions of MNF have been reported earlier to implicate CB receptoractivity. Preincubation with the CB1 receptor inverse agonist AM251rendered C6 cells refractory to the growth-inhibitory effect of MNF,while inhibition of CB2 receptor with AM630 had minimal effect againstMNF signaling (FIG. 18C). These results indicate that theanti-proliferative action of MNF occurs through CB1 receptor signaling.A coincident change in cell morphology and nuclear condensation wasobserved in MNF-treated C6 glioma cells, consistent with apoptosis (FIG.18D).

MNF Reduces Tumor Growth In Vivo in a Rat C6 Glioma Xenograft Model.

To determine whether MNF might have a therapeutic effect in vivo, a ratC6 glioma xenograft model was developed in immune-deficient mice.Tumor-bearing female nude mice were treated intraperitoneally with MNFdaily for 19 days. Significant reduction in tumor volume was observed inMNF-treated animals compared with the vehicle-treated group (P<0.008;FIG. 19A). These studies were performed in a second independent cohortof mice, and showed similar results (see FIG. 19B for combined results).Tumors were excised after the last day of treatment and snap frozen inliquid nitrogen for subsequent analyses.

Determination of MNF Levels In Vivo.

At the completion of the study, the tumors from the MNF-treated animalswere assayed for the tissue concentrations of MNF. The results indicatedthat significant concentrations of MNF accumulated in the tumor tissues,141.0±52.5 ng/ml/g tissue (cohort 1, n=9) and 214.4±65.5 ng/ml/g tissue(cohort 2, n=7), demonstrating that systemically administered. MNFreaches the proposed therapeutic target.

MNF Alters Gene Expression Profiling in C6 Glioma Xenografts.

Global gene expression profiling by microarray analysis was performed toidentify groups of genes in C6 glioma xenografts whose expression wasaltered upon MNF treatment as compared to tumor-bearing vehicle-treatedmice. Six independent biological samples from two cohorts of animalswere used in each group. After normalization and processing of themicroarray data, genes were considered as differentially expressed ifthey showed an absolute zratio of 1.5 or more between MNF and vehicleand had been assigned an adjusted P value <0.05 and false discovery rate<0.3. Principal component analysis (PCA) revealed a discriminatingpattern of significantly altered gene expression between MNF and vehiclegroups (FIG. 20A). Computational analysis of the datasets derived fromthis study led to the identification of a number of cellcycle-associated GO terms, such as “DNA replication”, “Cell cycle”,“Mitosis” and “Cell division” that are likely involved in the control oftumor growth during MNF treatment (Table 8). The analysis of theoverrepresented GO terms upon MNF treatment in the xenografttranscriptome revealed significant negative regulation of celldivision-related genes together with those involved in control ofmetabolism of nucleic acids. Using parameterized analysis of gene setenrichment (PAGE), additional insight was provided into regulatedsignaling pathways and biological processes affected by MNF. From thecollection of more than 308 gene sets, there were 55 gene sets whoseexpression levels were significantly altered by MNF, with the majorityof the gene sets (48/55) being down-regulated (Table 10).

The intensity of this signature from the two cohorts of animals isrepresented in FIG. 20B and a partial list of 10 gene sets influencedmost by MNF treatment is depicted in Table 9. Among the genes ofinterest many were downregulated in the MNF group compared to thevehicle control, including matrix metalloproteinase (MMP)-11 and 14(FIG. 20C). Treatment with MNF also sensitized C6 glioma tumor xenograftto growth arrest via the down-regulation of Galnt3 and other cell cycleregulators, such as Ccna2, Cdkn3 and Bub1b (FIG. 20C). In addition,there was significant reduction in expression of molecular markers forglial brain tumors upon MNF treatment. On the other hand,apoptosis-associated transcripts such as Casp1, Casp11 and Casp12 wereupregulated by MNF (FIG. 20C). Quantitative RT-PCR analysis confirmedthat MNF decreased the expression of Bub1b, Cdkn3 Ccna2, Olig1, Sox4,and Galnt3 as compared to control (FIG. 20D), thus validating themicroarray data. Overall, these results indicate routes by which MNFmight negatively affect glioma growth and progression. Also, theseresults indicate biomarkers, which can be used to determine theefficiency of MNF treatment as well as glioma growth and progression ingeneral. Thus, these studies disclose methods of diagnosing, prognosingand determining the efficiency of MNF as well as other treatments oftumor growth and progression in general by using the disclosedbiomarkers as indicators.

TABLE 8 List of GOTerms influenced by MNF treatment of a C6 gliomaxenograft model Annotation GO Term Set 1 Set 2 Combined GO0006270 DNAreplication initiation −4.0283 −3.7775 −5.0248 GO0048015Phosphoinositide mediated −4.2781 −3.5441 −5.2545 signaling GO0004527Exonuclease activity −2.5829 −5.4622 −5.8765 GO0000775 Chromosomepericentric −6.7644 −3.5296 −6.3081 region GO0007051 Spindleorganization −4.6166 −5.2288 −6.7039 and biogenesis GO0006260 DNAreplication −4.7695 −6.0446 −7.3157 GO0007049 Cell cycle −5.1361 −8.0162−9.3510 GO0005634 Nucleus −2.1680 −9.9454 −9.4291 GO0007067 Mitosis−7.5266 −7.6563 −10.1574 GO0051301 Cell division −6.9059 −8.0673−10.1585 Z scores for ‘MNF_Crtl’ are shown. These studies were performedon two independent cohorts of mice, with both cohorts showing equivalentresults.

TABLE 9 List of gene sets influenced by MNF treatment of C6 gliomaxenograft model Size Name Z score P value Fdr 40HIPPOCAMPUS_DEVELOPMENT_POSTNATAL 10.3511 0.0020 0.0344 280TARTE_MATURE_PC 5.3293 0.0097 0.1073 41 HYPOPHYSECTOMY_RAT_DN 3.92940.0170 0.1483 30 HDACI_COLON_BUT12HRS_UP 3.6676 0.0035 0.0527 60CELL_CYCLE −7.4222  1.5E−10 1.73E−08 37 P21_P53_ANY_DN −7.4300 5.12E−094.31E−07 74 LE_MYELIN_UP −8.8777 6.34E−08 4.64E−06 24CROONQUIST_IL6_STARVE_UP −9.0059 1.87E−18 6.31E−16 65IDX_TSA_UP_CLUSTER3 −10.1593 3.15E−20 1.76E−17 79SERUM_FIBROBLAST_CELLCYCLE −10.6997 2.98E−20 2.51E−17 Size indicates thenumber of genes found in each gene set. These studies were performed ontwo independent cohorts of mice, with both cohorts showing equivalentresults.

TABLE 10 MNF_Control, cohort 1 MNF_Control, cohort 2 MNF_Control,combined cohorts PathwayName (Zscore) (P_value) (fdr) (Zscore) (P_value)(fdr) (Zscore) (P_value) (fdr) HIPPOCAMPUS_DEVELOPMENT_ 3.6731 0.02110.1456 10.3512 0.0020 0.0344 11.4209 0.0017 0.0282 POSTNATALTARTE_MATURE_PC 3.6636 0.0129 0.1088 5.3293 0.0097 0.1073 6.8026 0.00040.0085 HDACI_COLON_BUT12HRS_UP 5.3212 0.0002 0.0053 3.6676 0.0035 0.05275.8481 0.0000 0.0007 HYPOPHYSECTOMY_RAT_DN 3.6187 0.0459 0.2249 3.92940.0170 0.1483 5.3951 0.0008 0.0144 HDACI_COLON_TSABUT_UP 4.1465 0.00800.0751 3.4480 0.0042 0.0600 5.2313 0.0006 0.0109 HDACI_COLON_BUT16HRS_UP3.6723 0.0199 0.1426 3.4287 0.0072 0.0877 4.8024 0.0010 0.0176HDACI_COLON_BUT24HRS_UP 3.0864 0.0323 0.1878 3.3154 0.0119 0.1209 4.44920.0010 0.0169 ATRBRCAPATHWAY −1.8815 0.0406 0.2138 −2.9977 0.0000 0.0000−3.4407 0.0000 0.0002 GOLDRATH_HP −2.4433 0.0229 0.1518 −2.2875 0.04410.2766 −3.4750 0.0007 0.0141 BRENTANI_REPAIR −3.8568 0.0002 0.0052−2.9453 0.0101 0.1094 −4.2370 0.0002 0.0048 REN_E2F1_TARGETS −2.91500.0121 0.1044 −3.5319 0.0026 0.0414 −4.4393 0.0008 0.0145 MOREAUX_TACI_−2.9881 0.0064 0.0635 −3.8456 0.0007 0.0154 −4.9180 0.0000 0.0009HI_IN_PPC_UP LAMB_CYCLIN_D3_GLOCUS −2.8637 0.0360 0.2028 −4.3054 0.00000.0000 −5.0503 0.0000 0.0001 POD1_KO_UP −3.9363 0.0052 0.0553 −4.35650.0064 0.0812 −5.1416 0.0007 0.0129 MOREAUX_TACI_HI_ −4.3721 0.00000.0011 −3.2936 0.0020 0.0334 −5.1872 0.0000 0.0000 VS_LOW_DN E2F1_DNA_UP−3.6781 0.0008 0.0142 −4.4885 0.0004 0.0099 −5.4288 0.0000 0.0014SHEPARD_CRASH_AND_BURN_ −3.9994 0.0035 0.0413 −4.0540 0.0139 0.1315−5.5788 0.0009 0.0160 MUT_VS_WT_DN IRITANI_ADPROX_LYMPH −5.1468 0.00070.0132 −3.3728 0.0387 0.2504 −5.7142 0.0004 0.0075 SHEPARD_GENES_COMMON_−4.0300 0.0067 0.0656 −4.3867 0.0259 0.1927 −5.7440 0.0019 0.0308BW_CB_MO PEART_HISTONE_DN −2.5055 0.0408 0.2142 −5.2170 0.0002 0.0063−5.7817 0.0001 0.0017 SHEPARD_BMYB_ −4.3350 0.0022 0.0300 −4.7947 0.01000.1089 −6.0639 0.0002 0.0054 MORPHOLINO_DN DAC_FIBRO_DN −4.4476 0.00070.0127 −4.9523 0.0007 0.0142 −6.4794 0.0000 0.0002 BRCA_PROGNOSIS_NEG−3.2974 0.0377 0.2052 −5.4682 0.0000 0.0003 −6.5976 0.0000 0.0002MANALO_HYPOXIA_DN −3.2723 0.0019 0.0268 −6.1956 0.0000 0.0000 −6.71550.0000 0.0000 LEE_TCELLS2_UP −4.1051 0.0020 0.0282 −5.6671 0.0000 0.0006−6.9464 0.0000 0.0000 KENNY_WNT_UP −4.0135 0.0093 0.0858 −5.5586 0.00000.0000 −6.9481 0.0000 0.0000 STEMCELL_NEURAL_UP −6.0983 0.0000 0.0001−5.0064 0.0000 0.0010 −7.2833 0.0000 0.0000 SASAKI_ATL_UP −5.0963 0.00000.0010 −5.5299 0.0000 0.0005 −7.2897 0.0000 0.0000SASAKI_TCELL_LYMPHOMA_ −5.0963 0.0000 0.0010 −5.5299 0.0000 0.0005−7.2897 0.0000 0.0000 VS_CD4_UP DNA_REPLICATION_REACTOME −3.8683 0.00390.0450 −6.5700 0.0000 0.0000 −7.3185 0.0000 0.0000 VERNELL_PRB_CLSTR1−3.4691 0.0269 0.1692 −6.5636 0.0000 0.0000 −7.4144 0.0000 0.0000GAY_YY1_DN −7.6370 0.0000 0.0000 −4.7456 0.0016 0.0295 −7.4972 0.00000.0001 CANCER_UNDIFFERENCTIATED_ −4.8288 0.0001 0.0034 −6.2847 0.00000.0013 −7.7981 0.0000 0.0002 META_UP CELL_CYCLE_KEGG −4.1925 0.00080.0139 −7.0955 0.0000 0.0000 −7.8764 0.0000 0.0000 ADIP_DIFF_CLUSTERS−4.6054 0.0050 0.0547 −6.8850 0.0000 0.0006 −7.9684 0.0000 0.0005OLDAGE_DN −4.5391 0.0006 0.0115 −7.1043 0.0000 0.0001 −8.1220 0.00000.0000 MIDDLEAGE_DN −3.6696 0.0102 0.0919 −7.7600 0.0000 0.0000 −8.24570.0000 0.0000 TARTE_PLASMA_BLASTIC −4.8905 0.0002 0.0054 −6.7905 0.00000.0000 −8.3308 0.0000 0.0000 YU_CMYC_UP −4.4689 0.0049 0.0539 −7.37230.0000 0.0000 −8.4501 0.0000 0.0000 CMV_IE86_UP −6.1497 0.0000 0.0000−6.5954 0.0000 0.0000 −8.5032 0.0000 0.0000 HOFFMANN_BIVSBII_BI_TABLE2−5.1273 0.0001 0.0030 −7.7114 0.0000 0.0005 −8.5773 0.0000 0.0002CELL_CYCLE −5.0187 0.0000 0.0013 −7.4222 0.0000 0.0000 −8.6730 0.00000.0000 KAMMINGA_EZH2_TARGETS −5.3211 0.0000 0.0004 −7.2448 0.0000 0.0000−8.8163 0.0000 0.0000 P21_P53_ANY_DN −5.0275 0.0012 0.0186 −7.43000.0000 0.0000 −8.8530 0.0000 0.0000 PRMT5_KD_UP −9.3903 0.0000 0.0000−5.4620 0.0000 0.0010 −8.9362 0.0000 0.0000 CROONQUIST_IL6_RAS_DN−6.7147 0.0000 0.0000 −7.7040 0.0000 0.0000 −9.7453 0.0000 0.0000DOX_RESIST_GASTRIC_UP −5.8557 0.0000 0.0001 −8.6145 0.0000 0.0000−10.1840 0.0000 0.0000 LEE_TCELLS3_UP −7.2409 0.0000 0.0000 −8.03390.0000 0.0000 −10.3427 0.0000 0.0000 LI_FETAL_VS_WT_KIDNEY_DN −5.44470.0001 0.0024 −9.1715 0.0000 0.0000 −10.4609 0.0000 0.0000ZHAN_MM_CD138_PR_VS_REST −6.1404 0.0000 0.0000 −9.0080 0.0000 0.0000−10.7002 0.0000 0.0000 CROONQUIST_IL6_STARVE_UP −6.5055 0.0000 0.0000−9.0059 0.0000 0.0000 −10.7172 0.0000 0.0000 LE_MYELIN_UP −6.5477 0.00000.0009 −8.8777 0.0000 0.0000 −10.8010 0.0000 0.0000 IDX_TSA_UP_CLUSTER3−6.1057 0.0000 0.0008 −10.1593 0.0000 0.0000 −11.3388 0.0000 0.0000SERUM_FIBROBLAST_ −7.2985 0.0000 0.0000 −10.6997 0.0000 0.0000 −12.26680.0000 0.0000 CELLCYCLE

Oligodendrocyte transcription factor 1 (Olig1) has been identified as anovel glioblastoma marker with diagnostic and prognostic value.Moreover, SRY-box 4 (Sox4) is a transcription factor that has beenimplicated in the determination of the cell fate and in tumorigenesis.The fact that Olig1 and Sox4 mRNA levels were reduced in MNF-treated C6glioma tumors compared with the control group is consistent withdecreased activation of molecular pathways leading to gliomagenesis. Theinvolvement of Cdkn3, Bub1b and Olig-1 in gliomagenesis is established.Here, evidence is provided of a down-regulation in the expression levelsof these genes in rat C6 glioma tumour xenografts in response to MNF,indicating that MNF and related analogs represent a therapeutic strategyin the treatment of high-grade gliomas. MNF is readily transportedacross the blood-brain barrier and can accumulate in the rat brain.

This example demonstrates MNF-dependent chemoprevention in aglioblastoma xenograft model in the mouse and provides a mechanism forits anticancer action in vivo. Also, diagnostic markers and method ofdetermining the efficiency of tumor treatment were revealed.

Example 9 MNF Targets GPR55-Mediated Ligand Internalization and ImpairsCancer Cell Motility

This example demonstrates that MNF targets GPR55-mediated ligandinternalization and impairs cancer cell motility.

(R,R′)-4′-Methoxy-1-naphthylfenoterol (MNF) promotes growth inhibitionand apoptosis of human HepG2 hepatocarcinoma cells via cannabinoidreceptor (CBR) activation. The synthetic CB1R inverse agonist, AM251,has been shown to block the anti-mitogenic effect of MNF in these cells;however, AM251 is also an agonist of the recently deorphanized,lipid-sensing receptor, GPR55, whose upregulation contributes tocarcinogenesis. Here, the role of GPR55 in MNF signaling in human HepG2and PANC-1 cancer cell lines in culture was investigated, with a focuson internalization of the fluorescent ligand Tocrifluor 1117 (T1117),reorganization of actin cytoskeleton, and cell motility as measured byscratch assay. Results indicated that GPR55 knockdown by RNAinterference markedly reduced cellular uptake of T1117, a process thatwas sensitive to MNF inhibition. GPR55 internalization mediated by theatypical cannabinoid O-1602 was blocked by MNF in GPR55-expressingHEK293 cells. Pretreatment of HepG2 and PANC-1 cells with MNFsignificantly abrogated the induction of ERK1/2 phosphorylation inresponse to AM251, O-1602 and fetal bovine serum, known to containbioactive lipids. Moreover, MNF exerted a coordinated negativeregulation of AM251 and O-1602 inducible processes, including change incell morphology and migration using scratch wound healing assay. Thisstudy shows that MNF impairs GPR55-mediated signaling and hastherapeutic potential in the management of cancer by facilitating futureresearch on GPR55.

Accumulation of cAMP by beta2-adrenoreceptor (β2-AR) agonists has beenassociated both with a decrease and increase in the mitogenic responseof cancer cell lines in culture. The mechanisms that determine celltype-specificity of β2-AR-mediated antitumor activity are poorlyunderstood and raise questions about possible biased agonism of theβ2-AR, whereby the molecular structure and stereochemistry of theagonist and the cellular environment of the receptor stabilizes one ormore ligand-specific active conformations of β2-AR. The main consequenceof biased agonism at the β2-AR [and other G protein-coupled receptors]is the activation of multiple G-protein isoforms and modulation ofvarious downstream signal transduction pathways that can lead todramatic differences in biological outcomes.

(R,R′)-4′-methoxy-1-naphthylfenoterol (MNF) is an analog of fenoterolwith a 573-fold greater selectivity for the β2-adrenergic receptor(β2-AR) than β1-AR. It enhances cAMP accumulation with EC50 value of3.90 nM in human β2-AR-overexpressing cells and attenuates proliferationof 1321N1 astrocytoma cells with IC₅₀ value of 3.98 nM. In contrast to(R,R′)-fenoterol, MNF activates both Gas and Gαi proteins and potentlystimulates cardiomyocyte contractility, consistent with its role as aβ2-AR agonist. However, it is disclosed herein that MNF treatment of thehuman-derived HepG2 hepatocarcinoma cell line causes growth arrest andapoptosis via a β2-AR-independent route. The MNF response was found tobe insensitive to the β2-AR antagonist ICI 118,551, and U87MG cells,which lack β2-AR binding activity, were responsive to the antimitogeniceffects of MNF. The presence of the naphthyl moiety in MNF led us tospeculate that it may share structural similarities with other ligandsand, therefore, behave as a dually acting compound with unique affinityand selectivity profile.

Cannabinoid receptors (CBRs) are often co-expressed with β2-AR in manytissues and various cell types, and their propensity to heterodimerizedemonstrates the potential for crosstalk between the two receptors. Infact, CBRs can modulate β2-AR activity. The engagement of CBRs byendogenous and synthetic cannabinoid ligands results in the regulationof proliferation and apoptosis of cancer cells, including HepG2 cells.It is interesting that treatment with selective pharmacological inverseagonists of CBRs blocks the antiproliferative actions of MNF in HepG2cells, consistent with the potential role of CBRs in MNF signaling. Eventhough AM251 and its clinical analog, rimonabant (SR141716A;N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride), interact with CB1R as inverse agonists, there is growingbody of evidence to suggest that they also act as agonists for therecently deorphanized GPR55. GPR55 is a G protein-coupled receptor withlipid-sensing properties whose upregulation contributes to theaggressive behavior of various cancer types. A role for ERK/MAP kinasesignaling during microglial activation and the promotion of cancer cellproliferation by GPR55 has been proposed. AM251 also promotes neutrophilchemotaxis by acting as a GPR55 agonist. Thus, the actions of AM251 andrimonabant, which have been widely interpreted as being mediated byCB1R, may, in fact, include off-target effects of GPR55 by this class ofcompounds.

The current example was designed to investigate the contribution ofGPR55 in MNF actions in two human cancer cell lines in culture, HepG2and PANC-1 cells. Using Tocrifluor 1117 (T1117), a fluorescent ligandthat binds to endogenous GPR55, with low affinity for CB1R,pharmacodynamic studies were performed and the data indicate that MNFsignificantly delays T1117 incorporation via impairment in theinternalization/recycling of GPR55. Treatment with MNF also resulted inimpaired ligand-mediated activation of downstream GPR55 signalingpathways in both tumor cell lines and their cell migration using thewound-healing assay. The present data show that cell exposure to MNFleads to impairment in GPR55 signaling.

Materials and Methods

Materials.

(R,R′)-4′-methoxy-1-naphthylfenoterol (MNF) was synthesized as describedpreviously (Jozwiak et al., Bioorg. Med. Chem. 18: 728, 736, 2007 whichis hereby incorporated by reference in its entirety). Eagle's minimumessential medium, trypsin solution, phosphate-buffered saline (PBS),fetal bovine serum (FBS), 100× solutions of sodium pyruvate (100 mM),L-glutamine (200 mM), and penicillin/streptomycin (a mixture of 10,000units/ml penicillin and 10,000 μg/ml streptomycin) were obtained fromQuality Biological (Gaithersburg, Md., USA). WIN 55,212-2, AM251, andAM630 were purchased from Cayman Chemical (Ann Arbor, Mich.), whereas CP55,940, O-1602 and Tocrifluor T1117 were from Tocris Bioscience(Ellisville, Mo., USA).

Maintenance and Treatment of Cell Lines.

Human HepG2 hepatocarcinoma cells and human PANC-1 cells were purchasedfrom ATCC (Manassas, Va., USA). HepG2 cells were maintained in Eagle'sminimum essential medium supplemented with 1% L-glutamine, 1% sodiumpyruvate, 1% penicillin/streptomycin, and 10% FBS (Hyclone, Logan, Utah,USA). PANC-1 cells were cultured in phenol red-free Dulbecco's modifiedEagle's medium (DMEM) supplemented with 4.5 g/L glucose and 1.5 g/Lsodium bicarbonate together with glutamine, pyruvate,penicillin/streptomycin and 10% FBS. HEK293 cells stably expressing theHA-tagged human GPR55 (hGPR55-HEK293) were a gift of Maria Waldhoer(Medical University of Graz, Graz, Austria) (Henstridge et al., Br. J.Pharmacol. 160: 604-614, 2010). The cells were maintained in DMEM with4.5 g/L glucose supplemented with 10% FBS, 0.2 mg/ml G418, andpenicillin/streptomycin. All cell lines were maintained in culture at37° C. in 5% CO2, and the medium was replaced every 2-3 days.

Cellular Uptake of TocriFluor T1117, a Fluorescently Labeled GPR55Agonist

Cells were grown in 35-mm glass bottom culture dishes (MatTek Corp.,Ashland, Mass., USA) for 48 hours until reaching ˜70% confluence.Serum-depleted cells were incubated either with DMSO (vehicle, 0.1%),MNF (1 μM), or synthetic cannabinoid compounds (AM630, AM251, O-1602, CP55,940) for 30 minutes prior to the addition of the novel fluorescentdiarylpyrazole cannabinoid ligand, Tocrifluor T1117 (10-100 nM). Cellswere imaged with a Zeiss LSM 710 confocal microscope equipped with atemperature-controlled and humidified CO2 chamber and a definite focussystem. A 561 nm DPSS laser was used for the excitation of the 5-TAMRAconjugate. The time-series function of the Zeiss Zen software was usedto collect images with a 40×1.3 NA objective every 30 s for up to onehour, with all confocal settings remaining the same throughout thestudies. Still images, movies and fluorescent intensity quantitationwere obtained from these series using the Zeiss Zen software. Studieswere repeated at least two to three times.

Gene Silencing.

HepG2 cells were transfected with siRNA oligos (1.25 μg) against CB1R,CB2R, or GPR55 or a non-silencing siRNA control (Santa CruzBiotechnology, Santa Cruz, Calif.) for 48 hours using 10 μl of siRNATransfection Reagent (Santa Cruz Biotechnology) following themanufacturer's protocol. siRNAs have been validated to perform efficientknockdown with minimal off-target effects. Following 48 hours of siRNAtreatment, cells were washed with PBS, and maintained in serum-freemedium before the addition of T1117.

GPR55 Internalization Assay.

Endocytosis of GPR55 was observed following a previously describedprotocol with minor modifications (Henstridge et al., Br. J. Pharmacol.160: 604-614, 2010). Briefly, hGPR55-HEK293 cells were grown on Lab-TekII CC2 chamber slides (Thermo Scientific Nunc, Rochester, N.Y.) for 48hours in regular media and then were serum starved for 1 hour. Apreincubation with 1:1000 rabbit HA antibody (Covance, Md.) wasperformed in the presence of vehicle (0.1% DMSO) or 1 μM MNF inserum-free media for 45 minutes at 37° C. in the CO₂ incubator. Cellswere then washed extensively with PBS and treated with 5 μM 01602 inserum-free media for 20 minutes at 37° C. in the CO₂ incubator.Subsequently, cells were washed three times, fixed in fresh 3.7%paraformaldehyde in PBS (10 min), and incubated with anti-rabbit AlexaFluor 488 antibody (Molecular Probes, Eugene, Oreg.; 1:1000, 30minutes). Cells were washed and fixed for a second time prior topermeabilization with 0.2% Triton X-100 (5 minutes). Incubation withanti-rabbit Alexa Fluor 568 antibody (Molecular Probes; 1:1000, 30minutes) was carried out to determine the extent of internalizedGPR55-HA antibody complex. The cells were washed in PBS, nuclearcounterstaining was performed with DAPI (4′,6-diamidino-2-phenylindole)added to the Prolong Gold antifade mounting medium (Invitrogen) andcured for 24 hours at room temperature in the dark. Images were acquiredwith a Zeiss LSM 710 confocal microscope (Thornwood, N.Y.) using CarlZeiss LSM software.

Scratch Assays.

These assays were carried out essentially as described previously (Fioriet al., Endocrinology 150: 2551-2560, 2009). In brief, cells were seededin 12-well nontreated polystyrene cell culture plates with flat bottom(Greiner Bio-One, Monroe, N.C.). Once the cells became confluent, ascratch wound was made with a pipette tip and pictured immediately (time0). Cells were pretreated either with vehicle (DMSO, 0.1%) or thesynthetic GPR55 ligands AM251 (1 μM) or O-1602 (1 μM) for 30 minutesfollowed by the addition of MNF (1 μM) where indicated. Cell migrationwas examined at 12, 24, 36, 48 hours and 12, 18, 24, 48 hours afterscratch for the HepG2 and PANC-1 cells, respectively. Images of the samefield were taken every 3 hours to determine the rate of cell migration.Images were captured on an Axiovert 200 inverted microscope (Carl Zeiss,Thornwood, N.Y.) mounted with an AxioCam HRc digital camera (Carl Zeiss)and the measurement of scratch area was performed with ImageJ 1.46ssoftware (National Institutes of Health, Bethesda, Md.). Each study wasperformed in duplicate dishes and repeated at least twice.

Synthesis of 5′-TAMRA-3-phenylpropan-1-amine.

Ten nmoles of the NHS ester of 5′-TAMRA (Sigma-Aldrich, St-Louis, Mo.)was incubated with 20 nmoles of 3-phenylpropan-1-amine (Sigma-Aldrich)in 1 ml of 0.1M PBS, pH 8.0 for 4 hours. The solution was stream driedunder nitrogen and reconstituted in 500 μl of a 1:1 solution of 10 mMTris-HCl, pH 8.0 in ethanol. The formation of5′-TAMRA-3-phenylpropan-1-amine (TAMRA-PPA, structure shown in FIG. 28A)and absence of the NHS ester of 5′-TAMRA was confirmed by massspectrometry (FIGS. 28B, 28C). A stock solution of 10 mM of TAMRA-PPAwas prepared, aliquoted and stored at −20° C.

Western Blot Analysis.

For detection of intracellular signaling proteins, cells were lysed inradioimmunoprecipitation buffer containing EGTA and EDTA (BostonBioProducts, Ashland, Mass.). The lysis buffer contained a proteaseinhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail(Calbiochem, San Diego, Calif.). Equivalent amount of proteins (14 and54 μg/well for PANC-1 and HepG2 cells, respectively) were separated on 4to 12% precast gels (Invitrogen, Carlsbad, Calif.) usingSDS-polyacrylamide gel electrophoresis under reducing conditions andthen electrophoretically transferred onto polyvinylidene fluoridemembrane (Invitrogen). Western blots were performed according tostandard methods, which involved blocking in 5% non-fat milk andincubated with the antibody of interest, followed by incubation with asecondary antibody conjugated with the enzyme horseradish perodixase.The detection of immunoreactive bands was performed by chemiluminescenceusing the ECL Plus Western Blotting Detection System (GE Healthcare,Piscataway, N.J.). The quantification of bands was done by volumedensitometry by using ImageJ software. The rabbit polyclonal antibodiesagainst EGFR were obtained from Cell Signaling Technology, Inc.(Beverly, Mass.) and the monoclonal anti-Hsp90 was purchased from SantaCruz Biotechnology, Inc. (Santa Cruz, Calif.).

Effect of MNF on O-1602-Mediated Increase in Cell Signaling.

Serum-starved cells were pretreated in the absence or presence of 1 μMMNF for 10 minutes followed by a 10 minute incubation with 0, 2.5 and 10μM O-1602 or 10% FBS, after which the levels of total ERK2 andphosphorylated forms of ERK1/2 (pErk1/2, Thr202/Tyr204), were determinedby Western blotting technique. All primary antibodies were purchasedfrom Cell Signaling Technology and used at a dilution recommended by themanufacturer.

Statistical Analysis.

Prism 4 (GraphPad Software, Inc., La Jolla, Calif.) running on apersonal computer was used to perform all the statistical data analysis.

Results

A Role for the Deorphanized GPR55 in the Cellular Incorporation ofT1117.

The fluorescently labeled AM251 analog, T1117, acts as a ligand forGPR55 with low affinity for CB1R. To address the issue of sensitivityand specificity of T1117 incorporation, serum-depleted cells weremaintained on a confocal microscope stage equipped with atemperature-controlled and humidified CO₂ chamber. Studied herein werethe characteristics of T1117 incorporation in human HepG2 and PANC-1cells at 37° C. The amount of cellular T1117 levels increased in adose-dependent manner, with a maximal incorporation at 100 nM and ahalf-maximal effect at approximately 8 nM (FIGS. 21A, 21B). Simultaneousaddition of a 100× molar excess of unlabeled AM251 caused a significant18-minutes delay in the cellular accumulation of T1117 (FIGS. 21C, 21D).Similarly, dropping the temperature to 10° C. reduced the rate of T1117uptake. To establish whether the cellular incorporation of T1117required the presence of the AM251 moiety, HepG2 cells were incubatedfor up to 1 hour with equimolar amounts either of T1117(5′-TAMRA-(3-phenylpropan-1-amine)-labeled AM251) or5′-TAMRA-3-phenylpropan-1-amine (TAMRA-PPA). Under these conditions,there was no significant incorporation of fluorescence upon cellincubation with TAMRA-PPA (FIG. 29C). These results demonstrate thatcellular accumulation of T1117 is rapid and saturable, and initiatedthrough competitive binding to cell surface receptors.

To confirm the role of GPR55 in the cellular uptake of T1117, HepG2cells were incubated with siRNA oligos against CB1R, CB2R or GPR55 andthe non-silencing siRNA control for 48 hours, after which T1117incorporation was monitored (FIG. 24A, 24B). Studies showed selectivereduction in gene expression when using siRNA duplexes against theindicated targets. In HepG2 cells transfected with control siRNA, entryof T1117 was observed with half-maximum incorporation at ˜15 minutes(FIG. 22A). Silencing of GPR55 blocked T1117 uptake by more than 6-foldwhereas a significant 10-12 minutes delay occurred upon CB1R silencing,ultimately resulting in a 40% reduction in cellular T1117 levels (FIG.22B). However, in cells transfected with CB2R siRNA, the profile ofT1117 incorporation was comparable to that of non-silencingsiRNA-transfected cells. These results indicate that GPR55 plays a majorrole in the cellular entry of T1117 and that constitutive CB1R activitymay participate in this GPR55 function.

To independently confirm these observations, HepG2 cells were pretreatedwith selective inverse agonists/antagonists of CB1R and CB2R prior tothe addition of T1117. The CB2R inverse agonist, AM630, was largelyinactive while pretreatment with the CBR agonist, WIN 55,212-2, markedlyincreased the rate of T1117 accumulation (FIG. 22C). The potentsynthetic cannabinoid agonist CP 55,940 has been reported to block GPR55internalization in a heterologous expression system. Here, a 30-minutespretreatment with CP 55,940 (0.25 μM) clearly reduced T1117 uptake by2.5-fold in HepG2 cells (FIG. 24D). Similarly, cell treatment with theatypical cannabinoid O-1602 (0.25 μM) caused a 12.5-minutes delay incellular accumulation of T1117, resulting in a 47% reduction in theamount of T1117 incorporated (FIG. 22D).

MNF Inhibits Cellular Incorporation of T1117.

The effects of MNF on GPR55 signaling were initially investigated usingthe incorporation of T1117 as an in vitro model of GPR55-dependentactivity. Pretreatment of HepG2 cells with 1 μM AM251 for 30 minutesneither inhibited nor enhanced constitutive T1117 incorporation,suggesting the absence of negative or positive allosteric modulation(FIGS. 23A, 23B). This is in contrast to the effect of concomitantaddition of a 100×-excess of AM251 with T1117, which significantlyreduced cellular T1117 accumulation under the same assay conditions(FIG. 23C), possibly because of the lower specific activity of thefluorescent marker. When this study was performed in the presence of MNFalone, a ˜70% reduction was observed (FIGS. 23A, 23B). Moreover, theaddition of AM251 either before or after the 30-minutes treatment withMNF caused a further lowering in T1117 incorporation, with a ˜6.8-foldreduction as compared to vehicle-treated cells.

To investigate whether the effects of the compounds were unique to HepG2cells, similar studies were conducted in PANC-1 cells in culture.Semi-quantitative PCR analysis indicated the presence of CB1R, CB2R andGPR55 in these cells. The results indicated that MNF was a potentinhibitor of T1117 incorporation in PANC-1 cells (FIGS. 23C and 23D).Thus, it would appear that MNF blocked endocytosis of a GPR55 ligand.

Effect of MNF on GPR55 Internalization and Downstream Signaling.

The effect of MNF on ligand-induced GPR55 internalization was performedin HEK293 cells stably expressing HA-tagged GPR55. Using confocal laserscanning microscopy, GPR55 was found to be located largely at the plasmamembrane of unstimulated cells (FIG. 24A). Addition of O-1602 for 20minutes led to marked endocytosis of HA-tagged GPR55, which was blockedby pretreatment with MNF (FIG. 24B).

Additional events downstream of GPR55 internalization may be impaired bycell treatment with MNF, as the redistribution of ligand-bound receptorsfrom the cell surface to endosomal compartment differentially regulatesvarious signaling pathways and their associated biological outcomes.Indeed, spatio-temporal activation of extracellular signal-regulatedkinase (ERK)-MAP kinase plays a role in the dynamic control of complexcellular functions. Here, exposure of HepG2 cells to O-1602dose-dependently increased ERK phosphorylation and MNF pretreatmentabrogated O-1602 responsiveness (FIGS. 25A and 25B). PANC-1 cellsexhibited the same behavior as HepG2 cells, and displayed exquisitesensitivity to MNF with regard to O-1602-mediated ERK phosphorylation(FIGS. 25C and 25D). Similar findings were observed following cellstimulation with AM251 with and without MNF pretreatment.

Bioactive concentrations of endocannabinoids are present in fetal bovineserum (250-700 nM of 2-arachidonoylglycerol), which strongly influencemonocytes/macrophage responses. As shown in FIGS. 26A-26D, pretreatmentwith MNF potently inhibited serum-induced ERK phosphorylation in bothcell lines.

A Role of MNF in the Morphology and Motility of Tumor Cells.

To further study the role of MNF and GPR55 activation in HepG2 andPANC-1 cell biology, possible alteration in morphology was investigated.Cells with irregular appearance and long filipodia and lamellipodia wereobserved in response to AM251 and O-1602 stimulation (FIGS. 26A and 26B,white arrows). Pretreatment with MNF rendered the cells refractory tothe change in morphology induced by AM251 and O-1602 in both tumor celllines. As shown in FIG. 26C, treatment of HepG2 and PANC-1 cells withO-1602 led to higher EGFR levels when compared to vehicle-treated cells,and MNF blocked this effect (FIG. 26C, lane 4 vs. 3) and that of AM251.These findings are consistent with the idea that MNF conferredrefractoriness to GPR55 signaling.

A Wound-Healing Assay In Vitro was then Performed to Investigate theEffects of MNF on Cell Motility.

As shown in FIGS. 27A and 29A, MNF alone had no effect on the motilityof HepG2 cells at the concentration used throughout the study (1 μM).This is in contrast, however, to its significant inhibitory effecttoward AM251-mediated increase in cell motility (FIGS. 27A, 29A). Therelative wound surface area after 24-hour treatment of HepG2 cells witheach condition depicted in FIG. 27B. Similar to its effects in HepG2cells, MNF also produced significant decrease in AM251-induced motilityof PANC-1 cells, but did not alter the constitutive rate of gap filling(FIGS. 27C, 27D and 29B). When tested against O-1602, a celltype-selective effect was observed in the presence of MNF. Inparticular, the ability of MNF to inhibit the wound closure evoked byO-1602 in PANC-1 cells was absent in HepG2 cells (FIG. 29C), indicativeof a complex mode of antagonism.

Engagement of the ‘cannabinoid-like receptor’ GPR55 triggers a number ofsignaling cascades that promote cell proliferation, migration, survivaland oncogenesis. MNF displays a number of characteristics associatedwith selective attenuation in GPR55 signaling, including 1) delayedcellular entry of a fluorescent GPR55 ligand, 2) inhibition of theinternalization of the ligand-occupied GPR55, and 3) a significantreduction in GPR55 agonist efficacy with regard to a number ofbiological readouts.

In cellular assays, the low level of non-specific uptake of thefluorophore alone (5′-TAMRA-PPA) makes T1117 (5′-TAMRA-PPA-conjugatedAM251) suitable for in vivo imaging approaches aimed at assessingoccupancy and internalization of GPR55. The compound T1117 has beenshown previously to measure the distribution of GPR55 in small mousearteries. Here, employing the siRNA-based gene silencing method, it wasdetermined that GPR55 was a main molecule responsible for T1117 entry inintact cells. In human HepG2 cells, the presence of GPR55 and theclassical CB1R was evidenced by PCR and functional assays. Bothreceptors trigger distinct signaling pathways in endothelial cells, andit was thus not surprising to observe in our study that the silencing ofCB1R by siRNA limited the response mediated by GPR55 while cellstimulation with an agonist of CB1R (WIN 55,212-2) resulted in anincrease in GPR55 constitutive activity. Although GPR55 interactscooperatively with CB2R to influence inflammatory responses ofneutrophils, pharmacological inhibition and silencing of CB2R by siRNAfailed to impact on T1117 incorporation in HepG2 cells. Thus,CB1R-triggered mechanism appears to contribute is some extent to theconstitutive GPR55-mediated T1117 uptake. The propensity of CB1R to formfunctional heterodimers with various GPCRs explains some of the celltype-specific physiological responses of GPR55.

Analysis of the data revealed that MNF significantly delayed thecellular accumulation of T1117 in serum-depleted cells expressingendogenous levels of GPR55, indicative of a decrease in the bindingaffinity of T1117 to GPR55 and/or impairment in constitutive cellsurface GPR55 internalization and recycling pathways. Pretreatment withAM251 for 30 minutes potentiated the effect of MNF, consistent with anegative cumulative event. In this model, AM251-bound GPR55 complexeswere internalized and any residual cell surface GPR55 receptors weretargeted by MNF, making this GPCR inaccessible for efficient T1117binding and/or internalization. Alternatively, inhibition of CB1R byAM251 may have also contributed to the observed potency in MNFsignaling. The ability of CP 55,940 to block cellular entry of T1117 wasconsistent with its role as a GPR55 antagonist.

The stimulation of GPR55-expressing HEK-293 cells with the atypicalcannabinoid O-1602 triggered rapid internalization of GPR55 through aMNF-inhibitable mechanism, indicating that under the current assayconditions, the potency of MNF was not appreciably influenced by theconditions of overexpression. GPCR desensitization and internalizationrequires the participation of β-arrestin translocation to the activatedreceptor. Using a β-arrestin translocation assay in a transienttransfection format, AM251 and its clinical analog rimonabant exhibitpotent activity as GPR55 agonists, whereas CP 55,940 blocks theformation of β-arrestin/GPR55 complexes. The possibility exists that MNFprevents the recruitment of β-arrestin to the GPR55, thereby providing anegative impact on internalization and recycling of this GPCR afteragonist exposure. In addition to its role in the promotion of GPCRinternalization, β-arrestin is required for activation of downstreamsignaling (e.g., ERK activation). GPR55 is thought to bind predominantlyG-protein G13, where it promotes Rho-dependent signaling in endothelialcells. Additional events downstream of GPR55 include activation of ERKand Ca2+ release from internal stores. Here, in vitro exposure of HepG2and PANC-1 cells to AM251 or O-1602 resulted in rapid increase in ERKphosphorylation, a process that was inhibited by cell pretreatment withMNF. An explanation for the significant reduction in agonist-stimulatedincrease in ERK phosphorylation in response to MNF is that ligand-boundGPR55 stimulates ERK activity once the receptor is internalized, incontrast to an earlier study by Li and colleagues showing thatopioid-mediated ERK activation is not dependent on κ-opioid receptorinternalization (J. Biol. Chem. 274: 12087-12094, 1999). Alternatively,MNF may interact with a putative allosteric binding site on GPR55 andelicit negative allosteric modulation of GPR55 agonists. It isnoteworthy that an allosteric binding site has been reported at theCB1R. MNF may inhibit signaling downstream of GPR55 to disrupt thebinding of T1117, receptor internalization and induction of thesignaling cascade leading to ERK activation.

Another striking observation from the disclosed study was the similaritybetween the effect of MNF on basal and agonist-induced ERKphosphorylation and on biological readouts, including GPR55-dependentcellular morphology and cell motility. ERK has been found to coordinateand regulate cell migration by promoting lamellipodial leading edgemovement via phosphorylation of the WAVE2 regulatory complex. Here,treatment of HepG2 and PANC-1 cells with AM251 or O-1602 led tofillipodia extension, which was blocked by MNF pretreatment. Moreover,MNF elicited a significant reduction in the rate of wound closure forGPR55 agonists in HepG2 and PANC-1 cells using a scratch wound-healingassay.

This example indicates MNF reduced proliferation and increased apoptosisin human HepG2 hepatocellular carcinoma cells and PANC-1 pancreaticcancer cell line in culture. The role of GPR55 in MNF signaling in HepG2and PANC-1 cells was investigated with a focus on internalization of thefluorescent ligand Tocrifluor 1117 (T1117), reorganization of actincytoskeleton, and cell motility as measured by scratch assay. Resultsindicated that GPR55 knockdown by RNA interference markedly reducedcellular uptake of T1117, a process that was sensitive to MNFinhibition. GPR55 internalization mediated by the atypical cannabinoidO-1602 was blocked by MNF in GPR55-expressing HEK293 cells. Pretreatmentof HepG2 and PANC-1 cells with MNF significantly abrogated the inductionof ERK1/2 phosphorylation in response to AM251, O-1602 and fetal bovineserum, known to contain bioactive lipids. Moreover, MNF exerted acoordinated negative regulation of AM251 and O-1602 inducible processes,including change in cell morphology and migration, using scratch-woundhealing assay. Thus, these studies show for the first time that MNFimpairs GPR55-mediated signaling and has therapeutic use in themanagement of cancer.

Example 10 Treatment of a CB Receptor Activity-Regulated Tumor

This example describes a method that can be used to treat a tumor in ahuman subject by administration of a composition comprising fenoterol, afenoterol analogue or a combination thereof at a therapeuticallyeffective amount to reduce or inhibit on or more signs or symptomsassociated with the tumor, such as a glioblastoma or hepatocellularcarcinoma. Although particular methods, dosages, and modes ofadministrations are provided, one skilled in the art will appreciatethat variations can be made without substantially affecting thetreatment.

A subject with a glioblastoma or hepatocellular carcinoma is selectedbased upon clinical symptoms. A biological sample is isolated from thesubject and CB receptor expression, including GPR55, and β2-ARexpression are determined by microarray, Western blotting orhistological studies. A positive result indicates that the tumor may betreated by administration of fenoterol, a disclosed fenoterol analogueor a combination thereof. In one particular example, a tissue biopsy isobtained from a subject with a primary brain tumor. Expression of β2-ARand GPR55 is determined in the sample. The absence of β2-ARs and thepresence of GPR55 in the sample indicates that the primary brain tumorcan be treated by administration of a composition including (R,R′)-MNF.The presence of β2-ARs and the presence of GPR55 indicates the tumor canbe treated by (R,R′)-MNF or fenoterol (or both) or other fenoterolanalogue(s) known to stimulate β2-ARs activity. The compositionincluding the desired compounds is intraperitoneally administered to thesubject at a concentration of 30 mg/kg/day for the first 10 days and 50mg/kg/day for the remaining 32 days. Tumor growth is then assessed 7days, 14 days, 21 days, 30 days, and 42 days following treatment. In oneexample, the effectiveness of the treatment is determined by imagingmethods, including non-invasive, high-resolution modalities, such ascomputed tomography (CT) and especially magnetic resonance imaging(MRI). For example, contrast agent uptake is monitored to determine theeffectiveness of the treatment. A decrease in permeability to theblood-brain barrier marked by an at least twenty percent (20%) decreasein uptake of a contrast agent as compared to reference value or thatmeasured prior to treatment indicates the treatment is effective. Also,a twenty-percent (20%) reduction in tumor size as compared to tumor sizeprior to treatment is considered to be an effective treatment. In oneexample, the therapeutic effectiveness is determined by measuringexpression or activity of one or more molecules demonstrated herein tobe regulated by MNF (see for example, Table 10). In some examples, asubject is administered an intravenous formulation of MNF used with acGMP-produced (R,R′)-4-methoxynaphthylfenoterol (such as a cGMP-produced(R,R′)-4-methoxynaphthylfenoterol 2 Kg formulation). In some examples, asubject is administered an intravenous formulation of MNF at aconcentration ranging from 0.1 to 10 mg/kg for 4 days as a single agentor in combination with other fenoterol analogs or standard agents usedin cancer chemotherapy over a two week period as a continuous or pulsedtherapy. In some examples, a subject is administered orally a 25 mg/kgdose of MNF formulated as a single agent or as a combination of(R,R′)-MNF and other MNF stereoisomers or fenoterol stereoisomers on adaily basis for a certain period of time, such as 1 month, 2 months, 3months, 4 months, 5 months, 6 months followed by additional periods ifdesired, based upon regression of or inhibition of tumor growth.

Example 11 Use of Disclosed Compositions Including (R,R′)-MNF or(R,R′)-NF (or Both) as an Adjuvant Therapy

This example describes a method that can be used to reduce, prevent, orretard tumor growth in a human subject that has been treated for amalignant astrocytoma.

A subject with an astrocytoma is selected based upon clinical symptomsand determined to have an astrocytoma expressing CB-receptors. Theprimary form of treatment of the malignant astrocytoma is open surgery.For subjects that are not surgical candidates, either radiation orchemotherapy is used as the initial treatment. Following the initialtreatment, a subject is administered a pharmaceutical compositioncontaining (R,R′)-MNF and/or (R,R′)-NF orally daily for an indefiniteperiod of time. The reoccurrence of tumor growth is monitored by imagingmethods, including non-invasive, high-resolution modalities, such as CTand MRI.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method of treating liver cancer, comprising:administering to a subject having liver cancer regulated by cannabinoidreceptor (CB) activity a therapeutically effective amount of a compoundto reduce one or more symptoms associated with the liver cancer, whereinadministering the therapeutically effective amount of the compound tothe subject inhibits growth of liver cancer cells regulated by CBactivity, wherein the compound is

and wherein the compound is optically active.
 2. The method of claim 1,wherein the compound is (R,R′)-4′-methoxy-1-naphthylfenoterol (MNF),(R,S′)-MNF, or a combination thereof.
 3. The method of claim 1, whereinreducing one or more symptoms associated with the liver cancer comprisesreducing tumor growth, reducing metastasis of a tumor, or a combinationthereof.
 4. The method of claim 1, wherein the CB receptor is GPR55. 5.The method of claim 1, wherein the liver cancer is hepatocellularcarcinoma.
 6. The method of claim 1, further administering to thesubject an additional chemotherapeutic agent prior to, concurrent with,or subsequent to administering the compound.
 7. The method of claim 1,wherein administering the compound comprises administering apharmaceutical composition comprising the compound and apharmaceutically acceptable carrier.
 8. The method of claim 7, whereinthe pharmaceutical composition is an injectable fluid or oral dosageform.
 9. The method of claim 8, wherein the oral dosage form is a syrup,a solution, a suspension, a powder, a pill, a tablet, or a capsule. 10.The method of claim 9, wherein the oral dosage form contains from about1.0 to about 50 mg of the compound.
 11. The method of claim 10, whereinthe oral dosage form is a tablet and administering the compoundcomprises administering one tablet to the subject two to four times aday.
 12. The method of claim 7, wherein the therapeutically effectiveamount of the compound is within a range from about 0.001 mg/kg to about10 mg/kg body weight administered orally in single or divided doses. 13.The method of claim 7, wherein the pharmaceutical composition is aninjectable fluid and is administered parenterally.
 14. The method ofclaim 13, wherein the therapeutically effective amount of the compoundis from about 1 mg/kg to about 100 mg/kg body weight.